RESUMO
Remodeling of the bone marrow microenvironment in chronic inflammation and in aging reduces hematopoietic stem cell (HSC) function. To assess the mechanisms of this functional decline of HSC and find strategies to counteract it, we established a model in which the Sfrp1 gene was deleted in Osterix+ osteolineage cells (OS1Δ/Δ mice). HSC from these mice showed severely diminished repopulating activity with associated DNA damage, enriched expression of the reactive oxygen species pathway and reduced single-cell proliferation. Interestingly, not only was the protein level of Catenin beta-1 (bcatenin) elevated, but so was its association with the phosphorylated co-activator p300 in the nucleus. Since these two proteins play a key role in promotion of differentiation and senescence, we inhibited in vivo phosphorylation of p300 through PP2A-PR72/130 by administration of IQ-1 in OS1Δ/Δ mice. This treatment not only reduced the b-catenin/phosphop300 association, but also decreased nuclear p300. More importantly, in vivo IQ-1 treatment fully restored HSC repopulating activity of the OS1Δ/Δ mice. Our findings show that the osteoprogenitor Sfrp1 is essential for maintaining HSC function. Furthermore, pharmacological downregulation of the nuclear b-catenin/phospho-p300 association is a new strategy to restore poor HSC function.
Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Camundongos , Animais , Células-Tronco Hematopoéticas/metabolismo , Diferenciação Celular , Medula Óssea/metabolismo , Envelhecimento , Espécies Reativas de Oxigênio/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
BACKGROUND: Mutations in cKIT or PDGFRA are found in up to 90% of patients with gastrointestinal stromal tumors (GISTs). Previously, we described the design, validation, and clinical performance of a digital droplet (dd)PCR assay panel for the detection of imatinib-sensitive cKIT and PDFGRA mutations in circulating tumor (ct)DNA. In this study, we developed and validated a set of ddPCR assays for the detection of cKIT mutations mediating resistance to cKIT kinase inhibitors in ctDNA. In addition, we cross-validated these assays using next generation sequencing (NGS). METHODS: We designed and validated five new ddPCR assays to cover the most frequent cKIT mutations mediating imatinib resistance in GISTs. For the most abundant imatinib-resistance-mediating mutations in exon 17, a drop-off, probe-based assay was designed. Dilution series (of decreasing mutant (MUT) allele frequency spiked into wildtype DNA) were conducted to determine the limit of detection (LoD). Empty controls, single wildtype controls, and samples from healthy individuals were tested to assess specificity and limit of blank (LoB). For clinical validation, we measured cKIT mutations in three patients and validated results using NGS. RESULTS: Technical validation demonstrated good analytical sensitivity, with a LoD ranging between 0.006% and 0.16% and a LoB ranging from 2.5 to 6.7 MUT fragments/mL. When the ddPCR assays were applied to three patients, the abundance of ctDNA in serial plasma samples reflected the individual disease course, detected disease activity, and indicated resistance mutations before imaging indicated progression. Digital droplet PCR showed good correlation to NGS for individual mutations, with a higher sensitivity of detection. CONCLUSIONS: This set of ddPCR assays, together with our previous set of cKIT and PDGFRA mutations assays, allows for dynamic monitoring of cKIT and PDGFRA mutations during treatment. Together with NGS, the GIST ddPCR panel will complement imaging of GISTs for early response evaluation and early detection of relapse, and thus it might facilitate personalized decision-making.
Assuntos
Ácidos Nucleicos Livres , DNA Tumoral Circulante , Tumores do Estroma Gastrointestinal , Humanos , DNA Tumoral Circulante/genética , DNA/uso terapêutico , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/genética , Tumores do Estroma Gastrointestinal/patologia , Mesilato de Imatinib/farmacologia , Mesilato de Imatinib/uso terapêutico , Mutação , Recidiva Local de Neoplasia/genética , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-kit/genética , Receptores Proteína Tirosina Quinases/genética , Neoplasias Gastrointestinais/tratamento farmacológico , Neoplasias Gastrointestinais/genéticaRESUMO
Interleukin 1 beta (IL-1 beta) is a potent proinflammatory factor during viral infection. Its production is tightly controlled by transcription of Il1b dependent on the transcription factor NF-kappaB and subsequent processing of pro-IL-1 beta by an inflammasome. However, the sensors and mechanisms that facilitate RNA virus-induced production of IL-1 beta are not well defined. Here we report a dual role for the RNA helicase RIG-I in RNA virus-induced proinflammatory responses. Whereas RIG-I-mediated activation of NF-kappaB required the signaling adaptor MAVS and a complex of the adaptors CARD9 and Bcl-10, RIG-I also bound to the adaptor ASC to trigger caspase-1-dependent inflammasome activation by a mechanism independent of MAVS, CARD9 and the Nod-like receptor protein NLRP3. Our results identify the CARD9-Bcl-10 module as an essential component of the RIG-I-dependent proinflammatory response and establish RIG-I as a sensor able to activate the inflammasome in response to certain RNA viruses.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , RNA Helicases DEAD-box/metabolismo , Inflamação/fisiopatologia , Interleucina-1beta/metabolismo , Vírus de RNA/fisiologia , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas Adaptadoras de Sinalização CARD , Caspase 1/metabolismo , Linhagem Celular , Células Cultivadas , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , Vírus da Encefalomiocardite/imunologia , Vírus da Encefalomiocardite/fisiologia , Ativação Enzimática , Ensaio de Imunoadsorção Enzimática , Interações Hospedeiro-Patógeno , Humanos , Immunoblotting , Inflamação/imunologia , Inflamação/virologia , Helicase IFIH1 Induzida por Interferon , Camundongos , Camundongos Knockout , Modelos Biológicos , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/fisiopatologia , Infecções por Vírus de RNA/virologia , Vírus de RNA/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/fisiologia , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
INTRODUCTION: Targeting the cell cycle machinery represents a rational therapeutic approach in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). Despite substantial response rates, clinical use of the PLK inhibitor volasertib has been hampered by elevated side effects such as neutropenia and infections. OBJECTIVES: The primary objective was to analyse whether a reduced dose of volasertib was able to limit toxic effects on the healthy haematopoiesis while retaining its therapeutic effect. METHODS: Bone marrow mononuclear cells (BMMNCs) of patients with MDS/sAML (n = 73) and healthy controls (n = 28) were treated with volasertib (1 µM to 1 nM) or vehicle control. Short-term viability analysis was performed by flow cytometry after 72 hours. For long-term viability analysis, colony-forming capacity was assessed after 14 days. Protein expression of RIPK3 and MCL-1 was quantified via flow cytometry. RESULTS: Reduced dose levels of volasertib retained high cell death-inducing efficacy in primary human stem and progenitor cells of MDS/sAML patients without affecting healthy haematopoiesis in vitro. Interestingly, volasertib reduced colony-forming capacity and cell survival independent of clinical stage or mutational status. CONCLUSIONS: Volasertib offers a promising therapeutic approach in patients with adverse prognostic profile. RIPK3 and MCL-1 might be potential biomarkers for sensitivity to volasertib treatment.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Hematopoese/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Síndromes Mielodisplásicas/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pteridinas/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Proteínas de Ciclo Celular/metabolismo , Feminino , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Masculino , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossíntese , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Pteridinas/efeitos adversos , Proteína Serina-Treonina Quinases de Interação com Receptores/biossíntese , Quinase 1 Polo-LikeRESUMO
Nucleotide-binding and oligomerization domain (NOD)-like receptors constitute a first line of defense against invading bacteria. X-linked Inhibitor of Apoptosis (XIAP) is implicated in the control of bacterial infections, and mutations in XIAP are causally linked to immunodeficiency in X-linked lymphoproliferative syndrome type-2 (XLP-2). Here, we demonstrate that the RING domain of XIAP is essential for NOD2 signaling and that XIAP contributes to exacerbation of inflammation-induced hepatitis in experimental mice. We find that XIAP ubiquitylates RIPK2 and recruits the linear ubiquitin chain assembly complex (LUBAC) to NOD2. We further show that LUBAC activity is required for efficient NF-κB activation and secretion of proinflammatory cytokines after NOD2 stimulation. Remarkably, XLP-2-derived XIAP variants have impaired ubiquitin ligase activity, fail to ubiquitylate RIPK2, and cannot facilitate NOD2 signaling. We conclude that XIAP and LUBAC constitute essential ubiquitin ligases in NOD2-mediated inflammatory signaling and propose that deregulation of NOD2 signaling contributes to XLP-2 pathogenesis.
Assuntos
Imunidade Inata , Inflamação/imunologia , Proteína Adaptadora de Sinalização NOD2/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Adaptadora de Sinalização NOD2/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
This prospective trial aimed to investigate whether tumor-specific cKIT and PDGFRA mutations can be detected and quantified in circulating tumor (ct)DNA in patients with active GIST, and whether detection indicates disease activity. We included 25 patients with active disease and cKIT or PDGFRA mutations detected in tissue. Mutant ctDNA was detected in the peripheral blood plasma using allele-specific ligation (L-)PCR and droplet digital (d)PCR. CtDNA harboring tumor-specific cKIT or PDGFRA mutations was detected at least once in 16 out of 25 patients using L-PCR (64%) and in 20 out of 25 patients with dPCR (80%). Using dPCR, the absolute numbers of ctDNA fragments (DNA copies/ml) and the mutant allele frequency (MAF; in percent of wild-type control) strongly correlated with tumor size expressed as RECIST1.1 sum of diameter (SOD) in mm (ρ = 0.3719 and 0.408, respectively, p < 0.0001) and response status (ρ = 0.3939 and 0.392, respectively, p < 0.0001 and p < 0.001). Specificity of dPCR for detection of progression was 79.2% with a sensitivity of 55.2% and dPCR discriminated CR from active disease with a specificity of 96% and s sensitivity of 44.7%. With L-PCR, correlations of MAF with tumor size and response status were less prominent. Serial ctDNA measurement reflected individual disease courses over time. Targeted panel sequencing of four patients detected additional driver mutations in all cases and secondary resistance mutations in two cases. Thus, ctDNA indicates disease activity in patients with GIST and should be incorporated as companion biomarker in future prospective trials.
Assuntos
DNA Tumoral Circulante/genética , DNA de Neoplasias/genética , Tumores do Estroma Gastrointestinal/genética , Mutação , Proteínas Proto-Oncogênicas c-kit/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , DNA Tumoral Circulante/sangue , DNA de Neoplasias/sangue , Feminino , Tumores do Estroma Gastrointestinal/tratamento farmacológico , Tumores do Estroma Gastrointestinal/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Avaliação de Resultados em Cuidados de Saúde/métodos , Reação em Cadeia da Polimerase/métodos , Estudos Prospectivos , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
NKT cells represent a small subset of glycolipid-recognizing T cells that are heavily implicated in human allergic, autoimmune, and malignant diseases. In the thymus, precursor cells recognize self-glycolipids by virtue of their semi-invariant TCR, which triggers NKT cell lineage commitment and maturation. During their development, NKT cells are polarized into the NKT1, NKT2, and NKT17 subsets, defined through their cytokine-secretion patterns and the expression of key transcription factors. However, we have largely ignored how the differentiation into the NKT cell subsets is regulated. In this article, we describe the mRNA-binding Roquin-1 and -2 proteins as central regulators of murine NKT cell fate decisions. In the thymus, T cell-specific ablation of the Roquin paralogs leads to a dramatic expansion of NKT17 cells, whereas peripheral mature NKT cells are essentially absent. Roquin-1/2-deficient NKT17 cells show exaggerated lineage-specific expression of nearly all NKT17-defining proteins tested. We show through mixed bone marrow chimera experiments that NKT17 polarization is mediated through cell-intrinsic mechanisms early during NKT cell development. In contrast, the loss of peripheral NKT cells is due to cell-extrinsic factors. Surprisingly, Roquin paralog-deficient NKT cells are, in striking contrast to conventional T cells, compromised in their ability to secrete cytokines. Altogether, we show that Roquin paralogs regulate the development and function of NKT cell subsets in the thymus and periphery.
Assuntos
Diferenciação Celular/imunologia , Células T Matadoras Naturais/imunologia , Subpopulações de Linfócitos T/imunologia , Ubiquitina-Proteína Ligases/imunologia , Animais , Citometria de Fluxo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Maintaining immune tolerance requires the production of Foxp3-expressing regulatory T (Treg) cells in the thymus. Activation of NF-κB transcription factors is critically required for Treg cell development, partly via initiating Foxp3 expression. NF-κB activation is controlled by a negative feedback regulation through the ubiquitin editing enzyme A20, which reduces proinflammatory signaling in myeloid cells and B cells. In naive CD4+ T cells, A20 prevents kinase RIPK3-dependent necroptosis. Using mice deficient for A20 in T lineage cells, we show that thymic and peripheral Treg cell compartments are quantitatively enlarged because of a cell-intrinsic developmental advantage of A20-deficient thymic Treg differentiation. A20-deficient thymic Treg cells exhibit reduced dependence on IL-2 but unchanged rates of proliferation and apoptosis. Activation of the NF-κB transcription factor RelA was enhanced, whereas nuclear translocation of c-Rel was decreased in A20-deficient thymic Treg cells. Furthermore, we found that the increase in Treg cells in T cell-specific A20-deficient mice was already observed in CD4+ single-positive CD25+ GITR+ Foxp3- thymic Treg cell progenitors. Treg cell precursors expressed high levels of the tumor necrosis factor receptor superfamily molecule GITR, whose stimulation is closely linked to thymic Treg cell development. A20-deficient Treg cells efficiently suppressed effector T cell-mediated graft-versus-host disease after allogeneic hematopoietic stem cell transplantation, suggesting normal suppressive function. Holding thymic production of natural Treg cells in check, A20 thus integrates Treg cell activity and increased effector T cell survival into an efficient CD4+ T cell response.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T Reguladores/fisiologia , Timo/citologia , Timo/fisiologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Animais , Apoptose , Diferenciação Celular , Citometria de Fluxo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Proteína Relacionada a TNFR Induzida por Glucocorticoide/genética , Doença Enxerto-Hospedeiro/prevenção & controle , Interleucina-2/imunologia , Ativação Linfocitária , Camundongos , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-rel/genética , Transdução de Sinais , Transplante de Células-Tronco , Timo/imunologia , Fator de Transcrição RelA/genética , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/deficiênciaRESUMO
To evaluate the outcomes of salvage third autologous stem cell transplantation (ASCT) in patients with relapsed multiple myeloma. We analyzed 570 patients who had undergone a third ASCT between 1997 and 2010 (European Society for Blood and Marrow Transplantation data), of whom 482 patients underwent tandem ASCT and a third ASCT at first relapse (AARA group) and 88 patients underwent an upfront ASCT with second and third transplantations after subsequent relapses (ARARA group). With a median follow-up after salvage third ASCT of 61 months in the AARA group and 48 months in the ARARA group, the day +100 nonrelapse mortality in the 2 groups was 4% and 7%, the incidence of second primary malignancy was 6% and 7%, the median progression-free survival was 13 and 8 months, and median overall survival (OS) was 33 and 15 months. In the AARA group, according to the relapse-free interval (RFI) from the second ASCT, the median OS after the third ASCT was 17 months if the RFI was <18 months, 37 months if the RFI was between 18 and 36 months, and 64 months if the RFI was ≥36 months (P < .001). In the ARARA group, the median OS after the third ASCT was 7 months if the RFI was <6 months, 13 months if the RFI was between 6 and 18 months, and 27 months if the RFI was ≥18 months (P < .001). In a multivariate analysis of the AARA group, the favorable prognostic factor was an RFI after second ASCT of ≥18 months. Progressive disease and a Karnofsky Performance Status score of <70 at third ASCT were unfavorable factors. A salvage third ASCT is of value for patients with relapsed myeloma, particularly for those with a long duration of response and chemosensitive disease at the time of transplantation.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Mieloma Múltiplo/terapia , Terapia de Salvação/métodos , Condicionamento Pré-Transplante/métodos , Transplante Autólogo/métodos , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Resultado do TratamentoRESUMO
The NF-κB regulator A20 limits inflammation by providing negative feedback in myeloid cells and B cells. Functional lack of A20 has been linked to several inflammatory and autoimmune diseases. To define how A20 affects the functionality of T effector cells in a highly inflammatory environment, we performed conventional allogeneic hematopoietic stem cell transplantation (allo-HSCT) with A20-deficient CD4+ and CD8+ donor T cells in mice. Severity and mortality of graft-versus-host disease (GVHD) after allo-HSCT was drastically reduced in recipients transplanted with conventional doses of A20-deficient T cells. Consistently, we found that the A20-deficient donor T-cell compartment was strongly diminished at various timepoints after allo-HSCT. However, proportionally more A20-deficient donor T cells produced IFN-γ and systemic inflammation was elevated early after allo-HSCT. Consequently, increasing the dose of transplanted A20-deficient T cells reversed the original phenotype and resulted in enhanced GVHD mortality compared to recipients that received A20+/+ T cells. Still, A20-deficient T cells, activated either through T cell receptor-dependent or -independent mechanisms, were less viable than control A20+/+ T cells, highlighting that A20 balances both, T-cell activation and survival. Thus, our findings suggest that targeting A20 in T cells may allow to modulate T-cell-mediated inflammatory diseases like GVHD.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Linfócitos T/imunologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/imunologia , Animais , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante HomólogoRESUMO
Activation of the C-type lectin receptor Dectin-1 by ß-glucans triggers multiple signals within DCs that result in activation of innate immunity. While these mechanisms can potently prime CD8+ cytotoxic T-cell (CTL) responses without additional adjuvants, the Dectin-1 effector pathways that control CTL induction remain unclear. Here we demonstrate that Dectin-1-induced CTL cross-priming in mice does not require inflammasome activation but strictly depends on the adapter protein Card9 in vitro. In vivo, Dectin-1-mediated Card9 activation after vaccination drives both expansion and activation of Ag-specific CTLs, resulting in long-lasting CTL responses that are sufficient to protect mice from tumor challenge. This Dectin-1-induced antitumor immune response was independent of NK cell function and completely abrogated in Card9-deficient mice. Thus, our results demonstrate that Dectin-1-triggered Card9 signaling but not inflammasome activation can potently cross-prime Ag-specific CTLs, suggesting that this pathway would be a candidate for immunotherapy and vaccine development.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Lectinas Tipo C/metabolismo , Neoplasias/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Proteínas Adaptadoras de Sinalização CARD/deficiência , Proteínas Adaptadoras de Sinalização CARD/genética , Apresentação Cruzada , Imunidade Inata , Inflamassomos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/fisiopatologia , Transdução de Sinais , VacinaçãoRESUMO
The crucial dependence of chronic lymphocytic leukemia (CLL) cells on signals derived from the B cell receptor (BCR) has encouraged the development of new inhibitors, which interfere with BCR signaling and demonstrate clinical benefits in nearly all patients. In addition, signaling through Toll-like receptor (TLR) 9 of the innate immune system has been shown to further contribute to the activation of CLL cells. However, responses to TLR9 engagement are not uniform, but diametrically opposed with cell death in some patients and cell proliferation in others. We now provide evidence that heterogeneous responses to TLR agonists are related to differences in the ability of CLL cells to activate the BCR-associated kinase Syk. Notably, expression of ZAP-70 appears to be of crucial importance for TLR9-mediated activation of Syk. We show that the activation of Syk provides an antiapoptotic signal, which is independent of Mcl-1, Bcl-2, and Bcl-XL, but related to the degradation of the proapoptotic Bim. Mechanistically, TLR9-mediated antiapoptotic signals in ZAP-70-positive CLL trigger secretion of immunoglobulin M, which then serves as (auto-) antigen for a prosurvival BCR signal. Thus, our data show that single activation of the innate immune receptor TLR9 is sufficient to fully engage BCR signaling in ZAP-70-positive CLL, protecting malignant cells from apoptosis. We conclude that the integration of TLR signaling into an adaptive immune response can further promote survival of CLL cells and may contribute to the unfavorable prognosis of ZAP-70-positive CLL.
Assuntos
Imunidade Adaptativa , Imunidade Inata , Leucemia Linfocítica Crônica de Células B/imunologia , Proteína-Tirosina Quinase ZAP-70/imunologia , Linfócitos B/imunologia , Linfócitos B/patologia , Linhagem Celular , Sobrevivência Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Proteínas Tirosina Quinases/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Transdução de Sinais , Quinase Syk , Receptor Toll-Like 9/imunologiaRESUMO
BACKGROUND: Cell separators are routinely used to collect CD34+ blood stem cells in the context of customized stem cell transplantation procedures. The Spectra Optia (Terumo BCT) is a novel development of the precursor instrument, the Cobe Spectra (Terumo BCT). STUDY DESIGN AND METHODS: In this report, 146 autologous and 42 allogeneic donors undergoing apheresis on the Cobe Spectra using the mononuclear cell (MNC) program 4.7 or on the Spectra Optia using the new continuous mononuclear cell (cMNC) program 11.2 are compared. RESULTS: Viability of cells and collection efficacy within the apheresis products was comparable for autologous and allogeneic products collected with the MNC or cMNC method. However, we found a reduced duration of the apheresis procedure and lower hematocrit within the apheresis products when using the cMNC in autologous and allogeneic donors. Moreover, allogeneic donors collected substantially more CD34+ cells per kilogram of body weight when using the cMNC method. Differences in platelets before and after apheresis were substantially smaller in this cohort when compared to the cohort collected with the MNC method. Neutrophil and platelet engraftment after autologous or allogeneic transplantation with a product collected with the MNC procedure was comparable to a transplantation with a product processed according to the cMNC method. CONCLUSION: Comparison of the MNC (Cobe Spectra) and the cMNC (Spectra Optia) methods demonstrated an equal performance and outcome. However, advantages were present using the cMNC method with respect to apheresis duration and hematocrit within the apheresis product (autologous/allogeneic donors) and numbers of CD34+ cells collected, especially in allogeneic donors.
Assuntos
Remoção de Componentes Sanguíneos/métodos , Adulto , Idoso , Antígenos CD34/metabolismo , Doadores de Sangue , Feminino , Mobilização de Células-Tronco Hematopoéticas , Humanos , Leucócitos Mononucleares/citologia , Masculino , Pessoa de Meia-Idade , Células-Tronco de Sangue Periférico/citologia , Transplante Homólogo/métodos , Adulto JovemRESUMO
Sfrp2 is overexpressed in stromal cells which maintain hematopoietic stem cells (HSCs) during in vitro culture. We here showed, that coculture of hematopoetic cells with stromal cells with reduced expression of Sfrp2 increases the number lineage-negative Kit(+) Sca-1(+) (LSK) and progenitor cells in vitro. The LSK cells from these cocultures showed activation of canonical Wnt signaling, higher levels of Ki-67, BrdU incorporation, and the number of γH2A.X positive foci. Total repopulating activity of these cultures was, however, diminished, indicating loss of HSC. To extend these in vitro data, we modelled stress in vivo, i.e., by aging, or 5-FU treatment in Sfrp2(-) (/) (-) mice, or replicative stress in regeneration of HSCs in Sfrp2(-) (/) (-) recipients. In all three in vivo stress situations, we noted an increase of LSK cells, characterized by increased levels of ß-catenin and cyclin D1. In the transplantation experiments, the increase in LSK cells in primary recipients was subsequently associated with a progressive loss of HSCs in serial transplantations. Similar to the in vitro coculture stress, in vivo genotoxic stress in 5-FU-treated Sfrp2(-) (/) (-) mice increased cell cycle activity of LSK cells with higher levels of BrdU incorporation, increased expression of Ki-67, and canonical Wnt signaling. Importantly, as noted in vitro, increased cycling of LSKs in vivo was accompanied by a defective γH2A.X-dependent DNA damage response and depolarized localization of acetylated H4K16. Our experiments support the view that Sfrp2 expression in the niche is required to maintain the HSC pool by limiting stress-induced DNA damage and attenuating canonical Wnt-mediated HSC activation. Stem Cells 2016;34:2381-2392.
Assuntos
Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Membrana/deficiência , Regeneração , Nicho de Células-Tronco , Estresse Fisiológico , Envelhecimento/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Microambiente Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Técnicas de Cocultura , Dano ao DNA , Fluoruracila/farmacologia , Hematopoese/efeitos dos fármacos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Regeneração/efeitos dos fármacos , Nicho de Células-Tronco/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Células Estromais/efeitos dos fármacos , Células Estromais/metabolismoRESUMO
BACKGROUND: Plerixafor is predominantly used for patients mobilizing inadequate stem cell numbers for autologous transplantation after stimulation with granulocyte-colony-stimulating factor (G-CSF). STUDY DESIGN AND METHODS: We here report on 300 patients undergoing stem cell mobilization with G-CSF, among them 36 poor mobilizers (CD34+ cell counts < 50 × 106 /L blood) receiving G-CSF alone and 49 receiving G-CSF in combination with plerixafor for rescue intervention. Mobilization efficacy and short-time outcome after autologous stem cell transplantation were analyzed and compared in the respective subgroups. RESULTS: Out of 49 patients treated with plerixafor and G-CSF, 46 (94%) collected sufficient hematopoietic stem cell numbers although the number was clearly inferior in poor mobilizers. Compared to good mobilizers, viability of CD34+ cells analyzed after collection was slightly reduced in poor mobilizers independent of the application of plerixafor. A total of 232 patients underwent autologous stem cell transplantation, among them 26 poor mobilizers who received only G-CSF and 31 patients who received G-CSF in combination with plerixafor. Time until neutrophil engraftment was in median 1 day later in poor mobilizers irrespective of the application of plerixafor. Platelet engraftment was in median 2 days delayed in patients mobilized with G-CSF and plerixafor compared to 1 day in poor mobilizers treated with G-CSF only. Frequency of detected CD38+ CD138+ CD45- CD56+ plasma cells in the apheresis products of myeloma patients was comparable for all groups. CONCLUSION: Our data demonstrate that plerixafor is highly effective as rescue measurement after mobilization failure with G-CSF alone and short-term clinical outcome after stem cell transplantation is comparable.
Assuntos
Remoção de Componentes Sanguíneos , Mobilização de Células-Tronco Hematopoéticas/métodos , Compostos Heterocíclicos/administração & dosagem , Mieloma Múltiplo/terapia , Transplante de Células-Tronco de Sangue Periférico , Células-Tronco de Sangue Periférico , Autoenxertos , Benzilaminas , Ciclamos , Feminino , Fator Estimulador de Colônias de Granulócitos/administração & dosagem , Humanos , Masculino , Mieloma Múltiplo/sangue , Plasmócitos/metabolismo , Estudos RetrospectivosRESUMO
Graft-versus-host-disease (GVHD) is a severe complication of allogeneic hematopoietic cell transplantation (allo-HCT) characterized by the production of high levels of proinflammatory cytokines. Activated Janus kinases (JAKs) are required for T-effector cell responses in different inflammatory diseases, and their blockade could potently reduce acute GVHD. We observed that inhibition of JAK1/2 signaling resulted in reduced proliferation of effector T cells and suppression of proinflammatory cytokine production in response to alloantigen in mice. In vivo JAK 1/2 inhibition improved survival of mice developing acute GVHD and reduced histopathological GVHD grading, serum levels of proinflammatory cytokines, and expansion of alloreactive luc-transgenic T cells. Mechanistically, we could show that ruxolitinib impaired differentiation of CD4(+) T cells into IFN-γ- and IL17A-producing cells, and that both T-cell phenotypes are linked to GVHD. Conversely, ruxolitinib treatment in allo-HCT recipients increased FoxP3(+) regulatory T cells, which are linked to immunologic tolerance. Based on these results, we treated 6 patients with steroid-refractory GVHD with ruxolitinib. All patients responded with respect to clinical GVHD symptoms and serum levels of proinflammatory cytokines. In summary, ruxolitinib represents a novel targeted approach in GVHD by suppression of proinflammatory signaling that mediates tissue damage and by promotion of tolerogenic Treg cells.
Assuntos
Doença Enxerto-Hospedeiro/tratamento farmacológico , Janus Quinase 2/antagonistas & inibidores , Inibidores de Proteínas Quinases/uso terapêutico , Pirazóis/uso terapêutico , Animais , Incompatibilidade de Grupos Sanguíneos/complicações , Células Cultivadas , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Doença Enxerto-Hospedeiro/etiologia , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Terapia de Alvo Molecular , Nitrilas , Pirimidinas , Índice de Gravidade de Doença , Transplante Homólogo/efeitos adversos , Resultado do TratamentoRESUMO
Myc oncogenic transcription factors (c-Myc, N-Myc, and L-Myc) coordinate the control of cell growth, division, and metabolism. In cancer, Myc overexpression is often associated with aggressive disease, which is in part due to the destruction of select targets by the ubiquitin-proteasome system (eg, SCF(Skp2)-directed destruction of the Cdk inhibitor p27(Kip1)). We reasoned that Myc would also regulate SUMOylation, a related means of posttranslational modification of proteins, and that this circuit would play essential roles in Myc-dependent tumorigenesis. Here, we report marked increases in the expression of genes that encode regulators and components of the SUMOylation machinery in mouse and human Myc-driven lymphomas, resulting in hyper-SUMOylation in these tumors. Further, inhibition of SUMOylation by genetic means disables Myc-induced proliferation, triggering G2/M cell-cycle arrest, polyploidy, and apoptosis. Using genetically defined cell models and conditional expression systems, this response was shown to be Myc specific. Finally, in vivo loss-of-function and pharmacologic studies demonstrated that inhibition of SUMOylation provokes rapid regression of Myc-driven lymphoma. Thus, targeting SUMOylation represents an attractive therapeutic option for lymphomas with MYC involvement.
Assuntos
Linfoma de Células B/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ácidos Anacárdicos/farmacologia , Animais , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Análise por Conglomerados , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Linfoma de Células B/genética , Camundongos , Camundongos Transgênicos , Poliploidia , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais , Sumoilação/efeitos dos fármacos , Transcrição Gênica , Enzimas Ativadoras de Ubiquitina/genética , Enzimas Ativadoras de Ubiquitina/metabolismoRESUMO
Acute myeloid leukemia originates from leukemia-initiating cells that reside in the protective bone marrow niche. CXCR4/CXCL12 interaction is crucially involved in recruitment and retention of leukemia-initiating cells within this niche. Various drugs targeting this pathway have entered clinical trials. To evaluate CXCR4 imaging in acute myeloid leukemia, we first tested CXCR4 expression in patient-derived primary blasts. Flow cytometry revealed that high blast counts in patients with acute myeloid leukemia correlate with high CXCR4 expression. The wide range of CXCR4 surface expression in patients was reflected in cell lines of acute myeloid leukemia. Next, we evaluated the CXCR4-specific peptide Pentixafor by positron emission tomography imaging in mice harboring CXCR4 positive and CXCR4 negative leukemia xenografts, and in 10 patients with active disease. [(68)Ga]Pentixafor-positron emission tomography showed specific measurable disease in murine CXCR4 positive xenografts, but not when CXCR4 was knocked out with CRISPR/Cas9 gene editing. Five of 10 patients showed tracer uptake correlating well with leukemia infiltration assessed by magnetic resonance imaging. The mean maximal standard uptake value was significantly higher in visually CXCR4 positive patients compared to CXCR4 negative patients. In summary, in vivo molecular CXCR4 imaging by means of positron emission tomography is feasible in acute myeloid leukemia. These data provide a framework for future diagnostic and theranostic approaches targeting the CXCR4/CXCL12-defined leukemia-initiating cell niche.
Assuntos
Complexos de Coordenação , Expressão Gênica , Leucemia Mieloide Aguda/diagnóstico por imagem , Leucemia Mieloide Aguda/genética , Imagem Molecular , Peptídeos Cíclicos , Tomografia por Emissão de Pósitrons , Receptores CXCR4/genética , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Marcação de Genes , Humanos , Leucemia Mieloide Aguda/patologia , Imageamento por Ressonância Magnética/métodos , Imagem Molecular/métodos , Tomografia por Emissão de Pósitrons/métodos , Receptores CXCR4/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
NIPA (nuclear interaction partner of ALK) is an F-box-like protein that monitors the timing of mitotic entry. Constitutively active NIPA delays mitotic entry by preventing accumulation of nuclear cyclin B1. Here, we have investigated the consequences of Nipa inactivation by using a conditional knockout strategy. Nipa-deficient animals are viable but show a lower birth rate and reduced body weight. Furthermore, Nipa-deficient males are sterile owing to a block of spermatogenesis during meiotic prophase. Whereas Nipa-/- mouse embryonic fibroblasts show no severe phenotype, Nipa-/- spermatocytes arrest during stage IV of the epithelial cycle with subsequent TUNEL-positive apoptosis resulting from improper synapsis, defects in the repair of DNA double-stranded breaks and synaptonemal complex formation. Moreover, we show nuclear accumulation of cyclin B1 with a subsequent premature increase in G2/M kinase activity in Nipa-/- spermatocytes. Together, these results reveal a novel role for NIPA in meiosis.
Assuntos
Ciclo Celular/fisiologia , Meiose/fisiologia , Proteínas Nucleares/metabolismo , Animais , Ciclo Celular/genética , Ciclina B1/genética , Ciclina B1/metabolismo , Quebras de DNA de Cadeia Dupla , Citometria de Fluxo , Immunoblotting , Imuno-Histoquímica , Imunoprecipitação , Masculino , Meiose/genética , Camundongos , Camundongos Knockout , Camundongos Mutantes , Proteínas Nucleares/genética , RNA Interferente Pequeno/genética , Espermatócitos/metabolismoRESUMO
BACKGROUND: In several European countries, preparation of cellular products with open manufacturing systems as used for cryopreservation of peripheral blood stem cells (PBSCs) needs to be performed in a clean-room facility. However, this form of manufacturing is highly expensive and laborious. Thus, safe techniques providing improved efficacy regarding time and material, which are in accordance with legal requirements are highly desirable. STUDY DESIGN AND METHODS: We have developed, validated, and applied a simple method for cryopreservation of PBSCs within a functionally closed-bag system using the closed cryo freeze prep set. This process fulfills good manufacturing practice requirements and allows for the cryopreservation of PBSCs without a clean-room facility. In addition to cryopreservation of PBSCs, we have recently successfully modified our system for processing, portioning, and cryopreservation of allogeneic donor lymphocytes. RESULTS: Since 2010, cryopreservation of PBSCs using a closed-bag system has been performed in our facility on a routine basis and 210 patients and healthy donors have been included in this analysis. No significant reduction in viability of CD34+ cells and no process-related contamination were observed. Outcome of hematopoietic stem cell transplantation regarding time of engraftment and infectious complications is comparable to products manufactured in conventional clean-room facilities. CONCLUSION: Our data confirm that cryopreservation of PBSCs within a functionally closed-bag system is safe, effective, and economical. Furthermore, the system has the potential to be extended to other manufacturing processes of cellular products.