RESUMO
SR-BI binds various lipoproteins, including HDL, LDL as well as VLDL, and mediates selective cholesteryl ester (CE) uptake. HDL derived CE accumulates in cellular lipid droplets (LDs), which also store triacylglycerol (TAG). We hypothesized that SR-BI could significantly facilitate LD formation, in part, by directly transporting LDL derived neutral lipids (NL) such as CE and TAG into LDs without lipolysis and de novo lipid synthesis. SR-BI overexpression greatly increased LDL uptake and LD formation in stably transfected HeLa cells (SR-BI-HeLa). LDs isolated from SR-BI-HeLa contained 4- and 7-times more CE and TAG, respectively, than mock-transfected HeLa (Mock-HeLa). In contrast, LDL receptor overexpression in HeLa (LDLr-HeLa) greatly increased LDL uptake, degradation with moderate 1.5- and 2-fold increases of CE and TAG, respectively. Utilizing CE and TAG analogs, BODIPY-TAG (BP-TAG) and BODIPY-CE (BP-CE), for tracking LDL NL, we found that after initial binding of LDL to SR-BI-HeLa, apoB remained at the cell surface, while BP-CE and BP-TAG were sorted and simultaneously transported together to LDs. Both lipids demonstrated limited internalization to lysosomes or endoplasmic reticulum in SR-BI-HeLa. In LDLr-HeLa, NLs demonstrated clear lysosomal sequestration without their sorting to LDs. An inhibition of TAG and CE de novo synthesis by 90-95% only reduced TAG and CE LD content by 45-50%, and had little effect on BP-CE and BP-TAG transport to LDs in SR-BI HeLa. Furthermore, intravenous infusion of 1-2 mg of LDL increased liver LDs in normal (WT) but not in SR-BI KO mice. Mice transgenic for human SR-BI demonstrated higher liver LD accumulation than WT mice. Finally, Electro Spray Infusion Mass Spectrometry (ESI-MS) using deuterated d-CE found that LDs accumulated up to 40% of unmodified d-CE LDL. We conclude that SR-BI mediates LDL-induced LD formation in vitro and in vivo. In addition to cytosolic NL hydrolysis and de novo lipid synthesis, this process includes selective sorting and transport of LDL NL to LDs with limited lysosomal NL sequestration and the transport of LDL CE, and TAG directly to LDs independently of de novo synthesis.
Assuntos
Gotículas Lipídicas/metabolismo , Lipídeos/química , Lipoproteínas LDL/metabolismo , Receptores Depuradores Classe B/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Compostos de Boro/metabolismo , Ésteres do Colesterol/metabolismo , Coenzima A Ligases/antagonistas & inibidores , Coenzima A Ligases/metabolismo , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de LDL/metabolismo , Triazenos/farmacologia , Triglicerídeos/metabolismoRESUMO
Apolipoprotein B mRNA-editing enzyme catalytic subunit 3 (APOBEC-3) enzymes are cytidine deaminases that are broadly and constitutively expressed. They are often up-regulated during carcinogenesis and candidate genes for causing the major single-base substitution in cancer-associated DNA mutations. Moreover, APOBEC-3s are involved in host innate immunity against many viruses. However, how APOBEC-3 mutational activity is regulated in normal and pathological conditions remains largely unknown. Heat shock protein levels are often elevated in both carcinogenesis and viral infection and are associated with DNA mutations. Here, using mutational analyses of hepatitis B virus (HBV), we found that Hsp90 stimulates deamination activity of APOBEC-3G (A3G), A3B, and A3C during co-expression in human liver HepG2 cells. Hsp90 directly stimulated A3G deamination activity when the purified proteins were used in in vitro reactions. Hsp40, -60, and -70 also had variable stimulatory effects in the cellular assay, but not in vitro Sequencing analyses further demonstrated that Hsp90 increased both A3G cytosine mutation efficiency on HBV DNA and total HBV mutation frequency. In addition, Hsp90 shifted A3G's cytosine region selection in HBV DNA and increased A3G's 5' nucleoside preference for deoxycytidine (5'-CC). Furthermore, the Hsp90 inhibitor 17-N-allylamino-17-demethoxygeldanamycin dose dependently inhibited A3G and A3B mutational activity on HBV viral DNA. Hsp90 knockdown by siRNA or by Hsp90 active-site mutation also decreased A3G activity. These results indicate that heat shock proteins, in particular Hsp90, stimulate APOBEC-3-mediated DNA deamination activity, suggesting a potential physiological role in carcinogenesis and viral innate immunity.
Assuntos
Desaminase APOBEC-3G/metabolismo , Citidina Desaminase/metabolismo , DNA Viral/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Vírus da Hepatite B/metabolismo , Hepatócitos/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Desaminase APOBEC-3G/química , Desaminase APOBEC-3G/genética , Carcinogênese , Citidina/metabolismo , Citidina Desaminase/química , Citidina Desaminase/genética , Análise Mutacional de DNA , DNA Recombinante/química , DNA Recombinante/metabolismo , DNA Viral/química , Desaminação , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Proteínas de Choque Térmico HSP90/genética , Células Hep G2 , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatócitos/imunologia , Hepatócitos/virologia , Humanos , Imunidade Inata , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/genética , Mutagênese , Taxa de Mutação , Fragmentos de Peptídeos/agonistas , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Mutação Puntual , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
PURPOSE: Progressive malignancy is the leading cause of death after allogeneic hematopoietic stem-cell transplantation (alloHSCT). After alloHSCT, B-cell malignancies often are treated with unmanipulated donor lymphocyte infusions (DLIs) from the transplant donor. DLIs frequently are not effective at eradicating malignancy and often cause graft-versus-host disease, a potentially lethal immune response against normal recipient tissues. METHODS: We conducted a clinical trial of allogeneic T cells genetically engineered to express a chimeric antigen receptor (CAR) targeting the B-cell antigen CD19. Patients with B-cell malignancies that had progressed after alloHSCT received a single infusion of CAR T cells. No chemotherapy or other therapies were administered. The T cells were obtained from each recipient's alloHSCT donor. RESULTS: Eight of 20 treated patients obtained remission, which included six complete remissions (CRs) and two partial remissions. The response rate was highest for acute lymphoblastic leukemia, with four of five patients obtaining minimal residual disease-negative CR. Responses also occurred in chronic lymphocytic leukemia and lymphoma. The longest ongoing CR was more than 30 months in a patient with chronic lymphocytic leukemia. New-onset acute graft-versus-host disease after CAR T-cell infusion developed in none of the patients. Toxicities included fever, tachycardia, and hypotension. Peak blood CAR T-cell levels were higher in patients who obtained remissions than in those who did not. Programmed cell death protein-1 expression was significantly elevated on CAR T cells after infusion. Presence of blood B cells before CAR T-cell infusion was associated with higher postinfusion CAR T-cell levels. CONCLUSION: Allogeneic anti-CD19 CAR T cells can effectively treat B-cell malignancies that progress after alloHSCT. The findings point toward a future when antigen-specific T-cell therapies will play a central role in alloHSCT.
Assuntos
Antígenos CD19/imunologia , Transplante de Células-Tronco Hematopoéticas , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/cirurgia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/transplante , Quimeras de Transplante , Adulto , Idoso , Progressão da Doença , Feminino , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Leucemia de Células B/imunologia , Leucemia de Células B/cirurgia , Masculino , Pessoa de Meia-Idade , Indução de Remissão , Transplante HomólogoRESUMO
The bone marrow (BM) microenvironment of multiple myeloma (MM) is reported to play a role in the biology of disease. In this study, we found that the extracellular BM microenvironment in MM contains a unique miRNA signature detectable by miRNA microarray and quantitative real-time PCR, which is partially represented in the peripheral blood. Eleven miRNAs were significantly decreased in both BM and serum of MM patients in comparison with controls. Evaluation of these miRNAs in plasma of a separate cohort of MM patients and controls confirmed significantly aberrant levels of let-7a, let-7b, let-7i, miR-15b, miR-16, and miR-20a in both serum and plasma. We then studied the myeloma precursor diseases and found that a subset of the MM miRNAs exhibited aberrant expression in monoclonal gammopathy of undetermined significance and smoldering myeloma. miRNA analysis of enriched CD138(+) plasma cells from MM and monoclonal gammopathy of undetermined significance found that most of the validated MM BM signature miRNAs were significantly decreased in MM plasma cells. Gene expression profiling indicated that multiple targets of the decreased miRNAs found increased expression in MM plasma cells, including ATF2, HRAS, HDAC4, TGFB1, TGFBR1, and mitogen-activated protein kinases. The findings suggest that these miRNAs are detectable in aberrant levels in the peripheral blood of patients with plasma cell proliferation and may play a role in aberrant plasma cell proliferation and disease progression.
Assuntos
Medula Óssea/metabolismo , MicroRNAs/genética , Mieloma Múltiplo/genética , Microambiente Tumoral/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proliferação de Células/genética , Progressão da Doença , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Gamopatia Monoclonal de Significância Indeterminada/genética , Plasmócitos/metabolismo , Adulto JovemRESUMO
PURPOSE: We hypothesized that lymphoid-selective host conditioning and subsequent adoptive transfer of sirolimus-resistant allogeneic T cells (T-Rapa), when combined with high-dose sirolimus drug therapy in vivo, would safely achieve antitumor effects while avoiding GVHD. EXPERIMENTAL DESIGN: Patients (n = 10) with metastatic renal cell carcinoma (RCC) were accrued because this disease is relatively refractory to high-dose conditioning yet may respond to high-dose sirolimus. A 21-day outpatient regimen of weekly pentostatin (P; 4 mg/m(2)/dose) combined with daily, dose-adjusted cyclophosphamide (C; ≤200 mg/d) was designed to deplete and suppress host T cells. After PC conditioning, patients received matched sibling, T-cell-replete peripheral blood stem cell allografts, and high-dose sirolimus (serum trough target, 20-30 ng/mL). To augment graft-versus-tumor (GVT) effects, multiple T-Rapa donor lymphocyte infusions (DLI) were administered (days 0, 14, and 45 posttransplant), and sirolimus was discontinued early (day 60 posttransplant). RESULTS: PC conditioning depleted host T cells without neutropenia or infection and facilitated donor engraftment (10 of 10 cases). High-dose sirolimus therapy inhibited multiple T-Rapa DLI, as evidenced by stable mixed donor/host chimerism. No antitumor responses were detected by RECIST criteria and no significant classical acute GVHD was observed. CONCLUSIONS: Immune-selective PC conditioning represents a new approach to safely achieve alloengraftment without neutropenia. However, allogeneic T cells generated ex vivo in sirolimus are not resistant to the tolerance-inducing effects of in vivo sirolimus drug therapy, thereby cautioning against use of this intervention in patients with refractory cancer.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Efeito Enxerto vs Tumor/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Transplante de Células-Tronco de Sangue Periférico , Quimeras de Transplante , Condicionamento Pré-Transplante , Adulto , Idoso , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/terapia , Ciclofosfamida/administração & dosagem , Feminino , Humanos , Imunofenotipagem , Imunoterapia Adotiva , Depleção Linfocítica , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias/patologia , Pentostatina/administração & dosagem , Fenótipo , Sirolimo/administração & dosagem , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Condicionamento Pré-Transplante/métodos , Transplante Homólogo , Resultado do TratamentoRESUMO
Although there are now fewer allo-SCTs performed for CML, leukemic relapse post transplant remains a persistent problem. To better define clinical and biological parameters determining postrelapse outcome, we studied 59 patients with CML relapsing after HLA-identical sibling allo-SCT between 1993 and 2008. Eighteen (30.5%) were transplanted in advanced phase and 41 (69.5%) in chronic phase. With a median follow-up from relapse of 7.9 years, 5-year post relapse survival (PRS) was 62%. Multivariate analysis found disease status at transplant, time to diagnosis of relapse from transplant and pretransplant tyrosine kinase inhibitor (TKI) use as significant factors associated with PRS. Analysis of BCR-ABL transcript expression in the hematopoietic progenitor compartment was performed in 36 patients (22 relapsed, 8 non-relapsed and 6 TKI alone controls). Patients with BCR-ABL expression in their early hematopoietic stem cell compartment (Lineage(-)CD34(+)CD38(-)CD90(+)) had worse survival irrespective of the disease status. We conclude that disease status remains the strongest clinical prognostic factor for PRS in CML following allo-SCT. The persistence of BCR-ABL expression in the progenitor cell compartment in some patients after SCT emphasizes the need to target CML-leukemia stem cells.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Adulto , Aloenxertos , Intervalo Livre de Doença , Feminino , Seguimentos , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Masculino , Pessoa de Meia-Idade , Recidiva , Estudos Retrospectivos , Taxa de SobrevidaRESUMO
Flow cytometric (FC) enumeration of abnormal plasma cells (APCs) for diagnosis and prognostication of plasma cell dyscrasias (PCD) is challenging. We studied antigen expression in normal plasma cells (NPC) (N = 34) and APC in a series of unselected PCD (N = 59). NPC subpopulations often demonstrated CD19(-), CD20(+), CD45(-) or dim and CD56(+), an immunophenotype observed in PCD. However abnormal CD81 was only observed in APCs (APC detection sensitivity 95%; specificity 100%). We evaluated differences in antigen expression patterns among MGUS (N = 14), SMM (N = 35) and MM (N = 10), finding the combination of CD45 and CD56 helpful in differentiating MGUS from SMM and MM (p = 0.0002).
Assuntos
Medula Óssea/patologia , Gamopatia Monoclonal de Significância Indeterminada/diagnóstico , Mieloma Múltiplo/diagnóstico , Paraproteinemias/diagnóstico , Plasmócitos/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Medula Óssea/imunologia , Antígeno CD56/genética , Antígeno CD56/imunologia , Diferenciação Celular , Diagnóstico Diferencial , Feminino , Citometria de Fluxo , Expressão Gênica , Humanos , Imunofenotipagem , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Masculino , Pessoa de Meia-Idade , Gamopatia Monoclonal de Significância Indeterminada/imunologia , Gamopatia Monoclonal de Significância Indeterminada/patologia , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/patologia , Paraproteinemias/imunologia , Paraproteinemias/patologia , Plasmócitos/imunologia , Tetraspanina 28/genética , Tetraspanina 28/imunologiaRESUMO
Plerixafor (Mozobil) is a CXCR4 antagonist that rapidly mobilizes CD34(+) cells into circulation. Recently, plerixafor has been used as a single agent to mobilize peripheral blood stem cells for allogeneic hematopoietic cell transplantation. Although G-CSF mobilization is known to alter the phenotype and cytokine polarization of transplanted T cells, the effects of plerixafor mobilization on T cells have not been well characterized. In this study, we show that alterations in the T cell phenotype and cytokine gene expression profiles characteristic of G-CSF mobilization do not occur after mobilization with plerixafor. Compared with nonmobilized T cells, plerixafor-mobilized T cells had similar phenotype, mixed lymphocyte reactivity, and Foxp3 gene expression levels in CD4(+) T cells, and did not undergo a change in expression levels of 84 genes associated with Th1/Th2/Th3 pathways. In contrast with plerixafor, G-CSF mobilization decreased CD62L expression on both CD4 and CD8(+) T cells and altered expression levels of 16 cytokine-associated genes in CD3(+) T cells. To assess the clinical relevance of these findings, we explored a murine model of graft-versus-host disease in which transplant recipients received plerixafor or G-CSF mobilized allograft from MHC-matched, minor histocompatibility-mismatched donors; recipients of plerixafor mobilized peripheral blood stem cells had a significantly higher incidence of skin graft-versus-host disease compared with mice receiving G-CSF mobilized transplants (100 versus 50%, respectively, p = 0.02). These preclinical data show plerixafor, in contrast with G-CSF, does not alter the phenotype and cytokine polarization of T cells, which raises the possibility that T cell-mediated immune sequelae of allogeneic transplantation in humans may differ when donor allografts are mobilized with plerixafor compared with G-CSF.
Assuntos
Citocinas/genética , Regulação da Expressão Gênica/imunologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Compostos Heterocíclicos/farmacologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Animais , Antígenos CD/biossíntese , Antígenos CD/genética , Benzilaminas , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/transplante , Ciclamos , Citocinas/biossíntese , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Doença Enxerto-Hospedeiro/imunologia , Humanos , Imunofenotipagem , Teste de Cultura Mista de Linfócitos , Linfopoese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Quimera por Radiação , Receptores CXCR4/efeitos dos fármacos , Organismos Livres de Patógenos Específicos , Subpopulações de Linfócitos T/imunologiaRESUMO
The risk of graft-rejection after allogeneic hematopoietic cell transplantation using conventional cyclophosphamide-based conditioning is increased in patients with bone marrow failure syndromes (BMFS) who are heavily transfused and often HLA-alloimmunized. Fifty-six patients with BMFS underwent fludarabine-based reduced-intensity conditioning and allogeneic peripheral blood progenitor cell (PBPC) transplantation at a single institution. The conditioning regimen consisted of intravenous cyclophosphamide, fludarabine, and equine antithymocyte globulin. Graft-versus-host disease (GVHD) prophylaxis included cyclosporine A alone or in combination with either mycophenolate mofetil or methotrexate. To reduce the risk of graft-rejection/failure, unmanipulated G-CSF mobilized PBPCs obtained from an HLA-identical or single HLA-antigen mismatched relative were transplanted rather than donor bone marrow. Despite a high prevalence of pretransplant HLA-alloimmunization (41%) and a heavy prior transfusion burden, graft-failure did not occur with all patients having sustained donor lympho-hematopoietic engraftment. The cumulative incidence of grade II-IV acute-GVHD and chronic-GVHD was 51.8% and 72%, respectively; with 87.1% surviving at a median follow-up of 4.5 years. A multivariate analysis showed pretransplant alloimmunization and rapid donor T-cell engraftment (≥95% donor by day 30) were both significantly (P < 0.05) associated with the development of chronic-GVHD (adjusted HR 2.13 and 2.99, respectively). These data show fludarabine-based PBPC transplantation overcomes the risk of graft-failure in patients with BMFS, although rapid donor T-cell engraftment associated with this approach appears to increase the risk of chronic-GVHD. (Clinicaltrials.gov identifier: NCT00003838).
Assuntos
Rejeição de Enxerto/prevenção & controle , Doença Enxerto-Hospedeiro/prevenção & controle , Hemoglobinúria Paroxística/terapia , Transplante de Células-Tronco de Sangue Periférico , Linfócitos T , Condicionamento Pré-Transplante , Adulto , Idoso , Anemia Aplástica , Soro Antilinfocitário/administração & dosagem , Antineoplásicos/administração & dosagem , Doenças da Medula Óssea , Transtornos da Insuficiência da Medula Óssea , Criança , Doença Crônica , Ciclosporina/administração & dosagem , Feminino , Rejeição de Enxerto/etiologia , Rejeição de Enxerto/patologia , Doença Enxerto-Hospedeiro/etiologia , Doença Enxerto-Hospedeiro/patologia , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Hemoglobinúria Paroxística/patologia , Humanos , Imunossupressores/administração & dosagem , Masculino , Pessoa de Meia-Idade , Ácido Micofenólico/administração & dosagem , Ácido Micofenólico/análogos & derivados , Fatores de Risco , Terapia de Salvação , Fatores de Tempo , Transplante Homólogo , Vidarabina/administração & dosagem , Vidarabina/análogos & derivados , Adulto JovemRESUMO
Scavenger receptor class B type I (SR-BI) is a multi-ligand receptor that binds a variety of lipoproteins, including high density lipoprotein (HDL) and low density lipoprotein (LDL), but lipoprotein(a) [Lp(a)] has not been investigated as a possible ligand. Stable cell lines (HEK293 and HeLa) expressing human SR-BI were incubated with protein- or lipid-labeled Lp(a) to investigate SR-BI-dependent Lp(a) cell association. SR-BI expression enhanced the association of both (125)I- and Alexa Fluor-labeled protein from Lp(a). By confocal microscopy, SR-BI was also found to promote the internalization of fluorescent lipids (BODIPY-cholesteryl ester (CE)- and DiI-labeled) from Lp(a), and by immunocytochemistry the cellular internalization of apolipoprotein(a) and apolipoprotein B. When dual-labeled ((3)H-cholesteryl ether,(125)I-protein) Lp(a) was added to cells expressing SR-BI, there was a greater relative increase in lipid uptake over protein, indicating that SR-BI mediates selective lipid uptake from Lp(a). Compared with C57BL/6 control mice, transgenic mice overexpressing human SR-BI in liver were found to have increased plasma clearance of (3)H-CE-Lp(a), whereas mouse scavenger receptor class B type I knockout (Sr-b1-KO) mice had decreased plasma clearance (fractional catabolic rate: 0.63 ± 0.08/day, 1.64 ± 0.62/day, and 4.64 ± 0.40/day for Sr-b1-KO, C57BL/6, and human scavenger receptor class B type I transgenic mice, respectively). We conclude that Lp(a) is a novel ligand for SR-BI and that SR-BI mediates selective uptake of Lp(a)-associated lipids.
Assuntos
Antígenos CD36/metabolismo , Lipoproteína(a)/metabolismo , Animais , Células HEK293 , Humanos , Lipoproteína(a)/sangue , Camundongos , Transporte ProteicoRESUMO
Human erythropoiesis is a dynamic and complex multistep process involving differentiation of early erythroid progenitors into enucleated RBCs. The mechanisms underlying erythropoiesis still remain incompletely understood. We previously demonstrated that erythropoietin-stimulated clone-1, which is selectively expressed in normal human erythroid-lineage cells, shares 99.5% identity with malignant fibrous histiocytoma-amplified sequences with leucine-rich tandem repeats 1 (MASL1). In this study, we hypothesized that the MASL1 gene plays a role in erythroid differentiation, and used a human erythroid cell culture system to explore this concept. MASL1 mRNA and protein expression levels were significantly increased during the erythroid differentiation of CD34(+) cells following erythropoietin (EPO) treatment. Conversely, MASL1 knockdown reduced erythroid differentiation in EPO-treated CD34(+) cells. In addition, MASL1 knockdown interrupted the Raf/MEK/ERK signaling pathway in CD34(+) cells. MASL1 mutant-transfected CD34(+) cells also showed decreased erythroid differentiation. Furthermore, inhibition of the SH3 domain of Son of Sevenless, which is an upstream adapter protein in EPO-induced erythroid differentiation, also reduced MASL1 expression and phosphorylation of Raf/MEK/ERK kinases that consequently reduced erythroid differentiation of EPO-induced CD34(+) cells. Importantly, we also demonstrated that MASL1 interacts physically with Raf1. Taken together, our data provide novel insights into MASL1 regulation of erythropoiesis through the Raf/MEK/ERK pathway.
Assuntos
Antígenos CD34/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Eritroides/citologia , Eritropoese , Eritropoetina/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Oncogênicas/metabolismo , Quinases raf/metabolismo , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/genética , Proliferação de Células , Células Cultivadas , Proteínas de Ligação a DNA/genética , Eritrócitos/citologia , Eritrócitos/metabolismo , Células Eritroides/metabolismo , Células Precursoras Eritroides/citologia , Células Precursoras Eritroides/metabolismo , Técnicas de Silenciamento de Genes , Granulócitos/citologia , Granulócitos/metabolismo , Humanos , Proteínas Oncogênicas/genética , RNA Mensageiro/genética , Proteínas Son Of Sevenless/metabolismo , Regulação para CimaAssuntos
Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Síndromes Mielodisplásicas/etiologia , Adulto , Neoplasias Ósseas/terapia , Feminino , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/terapia , Sarcoma de Ewing/terapia , Transplante Homólogo , Resultado do TratamentoRESUMO
Class B scavenger receptors (SR-Bs), such as SR-BI/II or CD36, bind lipoproteins but also mediate bacterial recognition and phagocytosis. In evaluating whether blocking receptors can prevent intracellular bacterial proliferation, phagocyte cytotoxicity, and proinflammatory signaling in bacterial infection/sepsis, we found that SR-BI/II- or CD36-deficient phagocytes are characterized by a reduced intracellular bacterial survival and a lower cytokine response and were protected from bacterial cytotoxicity in the presence of antibiotics. Mice deficient in either SR-BI/II or CD36 are protected from antibiotic-treated cecal ligation and puncture (CLP)-induced sepsis, with greatly increased peritoneal granulocytic phagocyte survival (8-fold), a drastic diminution in peritoneal bacteria counts, and a 50-70% reduction in systemic inflammation (serum levels of IL-6, TNF-α, and IL-10) and organ damage relative to CLP in wild-type mice. The survival rate of CD36-deficient mice after CLP was 58% compared with 17% in control mice. When compensated for mineralocorticoid and glucocorticoid deficiency, SR-BI/II-deficient mice had nearly a 50% survival rate versus 5% in mineralo-/glucocorticoid-treated controls. Targeting SR-B receptors with L-37pA, a peptide that functions as an antagonist of SR-BI/II and CD36 receptors, also increased peritoneal granulocyte counts, as well as reduced peritoneal bacteria and bacterium-induced cytokine secretion. In the CLP mouse sepsis model, L-37pA improved survival from 6 to 27%, reduced multiple organ damage, and improved kidney function. These results demonstrate that the reduction of both SR-BI/II- and CD36-dependent bacterial invasion and inflammatory response in the presence of antibiotic treatment results in granulocyte survival and local bacterial containment, as well as reduces systemic inflammation and organ damage and improves animal survival during severe infections.
Assuntos
Antígenos CD36/imunologia , Receptores Depuradores Classe B/imunologia , Sepse/imunologia , Animais , Antígenos CD36/metabolismo , Modelos Animais de Doenças , Granulócitos/imunologia , Granulócitos/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Fagocitose/imunologia , Receptores Depuradores Classe B/antagonistas & inibidores , Sepse/patologiaRESUMO
APOBEC-3 proteins induce C-to-U hypermutations in the viral genome of various viruses and have broad antiviral activity. Generally, only a small proportion of viral genomes (<10(-)(2)) are hypermutated by APOBEC-3s, but often many cytidines (up to 40%) are converted into uridine. The mechanism of this unique selective hypermutation remains unknown. We found that rat APOBEC-1 overexpression had a hypermutation pattern similar to that of APOBEC-3s on its substrate apolipoprotein B (apoB) mRNA. Transient plasmid transfection of rat APOBEC-1 resulted in 0.4% and 1.8% hypermutations with apoB mRNA in HepG2 and McA7777 cells, respectively. The low frequency of hypermutated apoB mRNA targets was enriched by differential DNA denaturation PCR at 72-76 °C, with hypermutation levels increasing up to 67%. Up to 69.6% of cytidines in HepG2 and up to 75.5% of cytidines in McA7777 cells were converted into uridines in the hypermutated apoB mRNA. When rat APOBEC-1 was overexpressed by adenovirus, the hypermutation frequency of apoB mRNA increased from 0.4% to â¼20% and was readily detected by regular PCR. However, this higher expression efficiency only increased the frequency of hypermutation, not the number of affected cytidines in hypermutated targets. Rat APOBEC-1 hypermutation was modulated by cofactors and eliminated by an E181Q mutation, indicating the role of cofactors in hypermutation. The finding of an APOBEC-3 hypermutation pattern with rat APOBEC-1 suggests that cofactors could also be involved in APOBEC-3 hypermutation. Using hepatitis B virus hypermutation, we found that KSRP increased APOBEC-3C and APOBEC-3B hypermutation. These data show that, like rat APOBEC-1 hypermutation, cellular factors may play a regulatory role in APOBEC-3 hypermutation.
Assuntos
Apolipoproteínas B/genética , Citidina Desaminase/biossíntese , Mutação , Desaminase APOBEC-1 , Animais , Citidina/metabolismo , Citidina Desaminase/genética , Células Hep G2 , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Humanos , Plasmídeos , Ratos , Transfecção , Uridina/biossínteseRESUMO
Donor lymphocyte infusion (DLI), a standard relapse treatment after allogeneic stem cell transplantation (AlloSCT), has limited efficacy and often triggers GVHD. We hypothesized that after AlloSCT tumor-infiltrating donor lymphocytes could be costimulated ex vivo to preferentially activate/expand antitumor effectors. We tested the feasibility and safety of costimulated, tumor-derived donor lymphocyte (TDL) infusion in a phase 1 trial. Tumor was resected from 8 patients with B-cell malignancy progression post-AlloSCT; tumor cell suspensions were costimulated with anti-CD3/anti-CD28 Ab-coated magnetic beads and cultured to generate TDL products for each patient. Costimulation yielded increased proportions of T-bet(+)FoxP3(-) type 1 effector donor T cells. A median of 2.04 × 10(7) TDL/kg was infused; TDLs were well tolerated, notably without GVHD. Two transient positron emission tomography (PET) responses and 2 mixed responses were observed in these refractory tumors. TDL are a feasible, tolerable, and novel donor cell therapy alternative for relapse after AlloSCT.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Doença de Hodgkin/cirurgia , Leucemia Linfocítica Crônica de Células B/cirurgia , Linfócitos do Interstício Tumoral/transplante , Linfoma Difuso de Grandes Células B/cirurgia , Humanos , Linfócitos do Interstício Tumoral/imunologia , Recidiva Local de Neoplasia/cirurgia , Transplante HomólogoRESUMO
Class B scavenger receptors (SR-B) are lipoprotein receptors that also mediate pathogen recognition, phagocytosis, and clearance as well as pathogen-induced signaling. In this study we report that three members of the SR-B family, namely, CLA-1, CLA-2, and CD36, mediate recognition of bacteria not only through interaction with cell wall LPS but also with cytosolic chaperonin 60. HeLa cells stably transfected with any of these SR-Bs demonstrated markedly (3- to 5-fold) increased binding and endocytosis of Escherichia coli, LPS, and chaperonin 60 (GroEL) as revealed by both FACS analysis and confocal microscopy imaging. Increased pathogen (E. coli, LPS, and GroEL) binding to SR-Bs was also associated with the dose-dependent stimulation of cytokine secretion in the order of CD36 > CLA-2 > CLA-1 in HEK293 cells. Pathogen-induced IL-6-secretion was reduced in macrophages from CD36- and SR-BI/II-null mice by 40-50 and 30-40%, respectively. Intravenous GroEL administration increased plasma IL-6 and CXCL1 levels in mice. The cytokine responses were 40-60% lower in CD36(-/-) relative to wild-type mice, whereas increased cytokine responses were found in SR-BI/II(-/-) mice. While investigating the discrepancy of in vitro versus in vivo data in SR-BI/II deficiency, SR-BI/II(-/-) mice were found to respond to GroEL administration without increases in either plasma corticosterone or aldosterone as normally seen in wild-type mice. SR-BI/II(-/-) mice with mineralocorticoid replacement demonstrated an â¼40-50% reduction in CXCL1 and IL-6 responses. These results demonstrate that, by recognizing and mediating inflammatory signaling of both bacterial cell wall LPS and cytosolic GroEL, all three SR-B family members play important roles in innate immunity and host defense.
Assuntos
Bactérias/imunologia , Antígenos CD36/imunologia , Inflamação/imunologia , Receptores Depuradores Classe B/imunologia , Transdução de Sinais/imunologia , Animais , Chaperonina 60/imunologia , Chaperonina 60/farmacologia , Citocinas/metabolismo , Escherichia coli/imunologia , Células HeLa , Humanos , Imunidade Inata , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Camundongos , Receptores Depuradores Classe B/deficiênciaRESUMO
We performed nonmyeloablative HSCT in 6 patients with a newly described genetic immunodeficiency syndrome caused by mutations in GATA2-a disease characterized by nontuberculous mycobacterial infection, monocytopenia, B- and NK-cell deficiency, and the propensity to transform to myelodysplastic syndrome/acute myelogenous leukemia. Two patients received peripheral blood stem cells (PBSCs) from matched-related donors, 2 received PBSCs from matched-unrelated donors, and 2 received stem cells from umbilical cord blood (UCB) donors. Recipients of matched-related and -unrelated donors received fludarabine and 200 cGy of total body irradiation (TBI); UCB recipients received cyclophosphamide in addition to fludarabine and TBI as conditioning. All patients received tacrolimus and sirolimus posttransplantation. Five patients were alive at a median follow-up of 17.4 months (range, 10-25). All patients achieved high levels of donor engraftment in the hematopoietic compartments that were deficient pretransplantation. Adverse events consisted of delayed engraftment in the recipient of a single UCB, GVHD in 4 patients, and immune-mediated pancytopenia and nephrotic syndrome in the recipient of a double UCB transplantation. Nonmyeloablative HSCT in GATA2 deficiency results in reconstitution of the severely deficient monocyte, B-cell, and NK-cell populations and reversal of the clinical phenotype. Registered at www.clinicaltrials.gov as NCT00923364.
Assuntos
Fator de Transcrição GATA2/genética , Transplante de Células-Tronco Hematopoéticas/métodos , Síndromes Mielodisplásicas/terapia , Adolescente , Adulto , Criança , Feminino , Fator de Transcrição GATA2/deficiência , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , Síndromes Mielodisplásicas/sangue , Síndromes Mielodisplásicas/genética , Projetos Piloto , Condicionamento Pré-Transplante , Transplante Homólogo , Resultado do Tratamento , Adulto JovemRESUMO
IL-15 uses the heterotrimeric receptor IL-2/IL-15Rß and the γ chain shared with IL-2 and the cytokine-specific IL-15Rα. Although IL-15 shares actions with IL-2 that include activation of natural killer (NK) and CD8 T cells, IL-15 is not associated with capillary leak syndrome, activation-induced cell death, or with a major effect on the number of functional regulatory T cells. To prepare for human trials to determine whether IL-15 is superior to IL-2 in cancer therapy, recombinant human IL-15 (rhIL-15) was produced under current good manufacturing practices. A safety study in rhesus macaques was performed in 4 groups of 6 animals each that received vehicle diluent control or rhIL-15 at 10, 20, or 50 µg/kg/d IV for 12 days. The major toxicity was grade 3/4 transient neutropenia. Bone marrow examinations demonstrated increased marrow cellularity, including cells of the neutrophil series. Furthermore, neutrophils were observed in sinusoids of enlarged livers and spleens, suggesting that IL-15 mediated neutrophil redistribution from the circulation to tissues. The observation that IL-15 administration was associated with increased numbers of circulating NK and CD8 central and effector-memory T cells, in conjunction with efficacy studies in murine tumor models, supports the use of multiple daily infusions of rhIL-15 in patients with metastatic malignancies.
Assuntos
Interleucina-15/toxicidade , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/imunologia , Antineoplásicos/farmacocinética , Antineoplásicos/toxicidade , Coagulação Sanguínea/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Humanos , Imunoterapia , Infusões Intravenosas , Interleucina-15/administração & dosagem , Interleucina-15/imunologia , Interleucina-15/farmacocinética , Fígado/efeitos dos fármacos , Fígado/patologia , Macaca mulatta , Neoplasias/imunologia , Neoplasias/terapia , Neutropenia/sangue , Neutropenia/induzido quimicamente , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/toxicidadeRESUMO
BACKGROUND: We previously showed that vaccination with one dose of PR1 and WT1 peptides induces transient anti-leukemia immunity. We hypothesized that maintenance of a sustained anti-leukemia response may require frequent boost injections. DESIGN AND METHODS: Eight patients with myeloid malignancies were enrolled in this phase II study, and 6 completed 6 injections of PR1 and WT1 peptides in Montanide-adjuvant with GM-CSF, every two weeks. RESULTS: Both high- and low-avidity PR1 or WT1-specific CD8(+) T cells were detected in all evaluable patients after the first vaccine dose. Repeated vaccination led to selective deletion of high avidity PR1- and WT1-specific CD8(+) T cells and was not associated with significant reduction in WT1-expression. Additional boosting failed to increase vaccine-induced CD8(+) T-cell frequencies further and in all patients the response was lost before the 6(th) dose. PR1- or WT1-specific CD8(+) T cells were not detected in bone marrow samples, excluding their preferential localization to this site. Following a booster injection three months after the 6(th) vaccine dose, no high-avidity PR1 or WT1-specific CD8(+) T cells could be detected, whereas low-avidity T cells were readily expanded. CONCLUSIONS: These data support the immunogenicity of PR1 and WT1 peptide vaccines. However, repeated delivery of peptides with Montanide-adjuvant and GM-CSF leads to rapid loss of high-avidity peptide-specific CD8(+) T cells. These results may offer an explanation for the lack of correlation between immune and clinical responses observed in a number of clinical trials of peptide vaccination. New approaches are needed to induce long-term high-avidity memory responses against leukemia antigens.
Assuntos
Vacinas Anticâncer/uso terapêutico , Leucemia/terapia , Transtornos Mieloproliferativos/terapia , Vacinas de Subunidades Antigênicas/uso terapêutico , Proteínas WT1/uso terapêutico , Adjuvantes Imunológicos/administração & dosagem , Adulto , Idoso , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/síntese química , Vacinas Anticâncer/imunologia , Epitopos/imunologia , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Imunização , Leucemia/imunologia , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/imunologia , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/síntese química , Vacinas de Subunidades Antigênicas/imunologia , Proteínas WT1/administração & dosagem , Proteínas WT1/síntese química , Proteínas WT1/imunologiaRESUMO
BACKGROUND: The ability to expand virus- or tumor-specific T cells without damaging their functional capabilities is critical for success adoptive transfer immunotherapy of patients with opportunistic infection or tumor. Careful comparisons can help identify expansion methods better suited for particular clinical settings and identify recurrent deficiencies requiring new innovation. METHODS: We compared the efficacy of magnetic beads coated with anti-CD3 and anti-CD28 (anti-CD3/CD28 beads), and soluble anti-CD3 plus mixed mononuclear cells (designated a rapid expansion protocol or REP) in expanding normal human T cells. RESULTS: Both anti-CD3/CD28 beads and soluble anti-CD3 promoted extensive expansion. Beads stimulated greater CD4 cell growth (geometric mean of 56- versus 27-fold (p < 0.01) at day 21) but both stimulated similar CD8 expansion (189- versus 186-fold). Phenotypically, bead-treated CD4 and CD8 T cells and anti-CD3-treated CD4 cells typically assumed an effector/effector memory phenotype by day 14. By comparison, a subset of anti-CD3-treated CD8 cells, derived from naïve cells, retained much greater expression of CD45RA, CD27 and CCR7, than matched bead-treated cells despite comparable expansion. These cells were clearly distinguishable from CD45RA+ terminally differentiated effector cells by the presence of CD27, the absence of CD57 and their inability to produce cytokines after stimulation. When used to expand previously stimulated cells, anti-CD3 plus autologous MNCs produced much less antigen-induced cell death of CD8 cells and significantly more CD8 expansion than beads. CONCLUSIONS: Anti-CD3/CD28 beads are highly effective for expanding CD4 cells, but soluble anti-CD3 has significant potential advantages for expanding CD8 T cells, particularly where preservation of phenotypically "young" CD8 cells would be desirable, or where the T cells of interest have been antigen-stimulated in vitro or in vivo in the recent past.