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1.
Adv Exp Med Biol ; 1131: 1065-1078, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646545

RESUMO

Our blood serum Ca2+ levels are maintained within a narrow range (Ca2+ homeostasis) through a complex feedback system. However, local bone marrow Ca2+ levels can reach high concentrations, at least transiently, due to bone resorption, which is one of the notable features of the bone marrow stroma. Bone homeostasis is maintained by both the balance between osteoblastic bone formation and osteoclastic bone resorption and the balance of mesenchymal stem cell differentiation into osteoblasts and adipocytes. It has been reported that under culture conditions of infrequent adipocyte differentiation (no treatment with insulin or dexamethasone), high extracellular Ca2+ enhances osteoblast but not adipocyte accumulation in bone marrow stromal cells. In contrast, under culture conditions of predominant adipocyte differentiation (treatment with insulin and dexamethasone), high extracellular Ca2+ enhances adipocyte but not osteoblast accumulation in bone marrow stromal cells. Thus, the increased extracellular Ca2+ caused by bone resorption might enhance osteoblast development to reform missing bone under conditions of infrequent adipocyte differentiation (such as the normal physiological state) and might accelerate adipocyte accumulation instead of osteoblastic bone formation under conditions of predominant adipocyte differentiation (such as aging, obesity, use of glucocorticoids, and postmenopause). Moreover, increased adipocyte accumulation in bone marrow suppresses lymphohematopoiesis and contributes to a dysfunction of osteogenesis.


Assuntos
Medula Óssea , Cálcio , Células-Tronco Mesenquimais , Adipócitos/citologia , Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/patologia , Cálcio/metabolismo , Diferenciação Celular , Humanos , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia
3.
Adv Exp Med Biol ; 1143: 59-74, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31338815

RESUMO

One of the bottlenecks of the treatments for malignant hematopoietic disorders is the unavailability of sufficient amount of hematopoietic stem cells (HSCs). HSCs are considered to be originated from the aorta-gonad-mesonephros and gradually migrates into fetal liver and resides in a unique microenvironment/niche of bone marrow. Although many intrinsic and extrinsic factors (niche components) are reported to be involved in the origination, maturation, migration, and localization of HSCs at different developmental stages, the detailed molecular mechanisms still remain largely unknown. Previous studies have shown that intrinsic metabolic networks may be critical for the cell fate determinations of HSCs. For example, HSCs mainly utilize glycolysis as the main energy sources; oxidative phosphorylation is required for the homeostasis of HSCs; lipid or amino acid metabolisms may also sustain HSC stemness. Mechanistically, lots of regulatory pathways, such as MEIS1/HIF1A and PI3K/AKT/mTOR signaling, are found to fine-tune the different nutrient metabolisms and cell fate commitments of HSCs. However, more efforts are required for the optimization and establishment of precise metabolic techniques specific for the HSCs with relatively rare cell frequency and understanding of the basic metabolic properties and their underlying regulatory mechanisms of different nutrients (such as glucose) during the different developmental stages of HSCs.


Assuntos
Diferenciação Celular , Células-Tronco Hematopoéticas , Transdução de Sinais , Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos
4.
Life Sci ; 232: 116625, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31276691

RESUMO

AIMS: The chondrogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) is critical for cartilage regeneration. Tissues constructed from BMSCs through cartilage tissue engineering still exhibit some histological, morphological, and biomechanical differences from normal cartilage tissues. Cyclic tensile strain (CTS) can increase chondrogenic gene expression and reduce hypertrophic gene expression in chondrocytes. miR-365 has been identified as a mechanoresponsive microRNA and is an important regulator of both chondrocyte hypertrophy and differentiation. Therefore, we hypothesized that CTS may promote the chondrogenesis of BMSCs by upregulating the expression of miR-365. METHODS: BMSCs were subjected to CTS to investigate the effects and mechanism on chondrogenesis. An Agilent miRNA microarray was used to profile miRNAs in the CTS-treated BMSCs and 3D-cultured control BMSCs. miR-365 was shown to interact with HDAC4 mRNA through a luciferase reporter assay. An animal cartilage defect model was constructed and different groups of BMSCs were implanted to investigate their in vivo effect. KEY FINDINGS: CTS promoted BMSC chondrogenesis. miR-365 was significantly upregulated in CTS-treated cells and played an important role in CTS-mediated chondrogenesis. Luciferase assays showed that HDAC4 is a direct target of miR-365. An in vivo study showed that CTS treatment and miR-365 overexpression could promote cartilage regeneration from BMSCs. SIGNIFICANCE: CTS can promote the expression of miR-365, a crucial mechanosensitive microRNA involved in the chondrogenesis of BMSCs, which directly inhibits the expression of HDAC4, in turn, enhancing the chondrogenesis of BMSCs.


Assuntos
Condrogênese/genética , Células-Tronco Mesenquimais/fisiologia , MicroRNAs/genética , Animais , Medula Óssea/metabolismo , Células da Medula Óssea/citologia , Cartilagem/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Condrócitos/metabolismo , Condrogênese/fisiologia , MicroRNAs/metabolismo , Ratos , Transdução de Sinais , Resistência à Tração/fisiologia , Engenharia Tecidual
5.
Nat Methods ; 16(8): 695-698, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31308548

RESUMO

Single-cell RNA sequencing is often applied in study designs that include multiple individuals, conditions or tissues. To identify recurrent cell subpopulations in such heterogeneous collections, we developed Conos, an approach that relies on multiple plausible inter-sample mappings to construct a global graph connecting all measured cells. The graph enables identification of recurrent cell clusters and propagation of information between datasets in multi-sample or atlas-scale collections.


Assuntos
Medula Óssea/metabolismo , Biologia Computacional/métodos , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Célula Única/métodos , Humanos
6.
Ann Hematol ; 98(10): 2339-2346, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31250082

RESUMO

Calreticulin (CALR) mutations are detected in the majority of JAK2 wild type patients with essential thrombocythemia (ET). Unlike JAK2V617F and MPL point mutations, CALR mutations are highly heterogeneous, with several types of indels being reported so far. CAL2 is a monoclonal antibody specifically recognizing the C-neoterminal peptide derived from all the frameshift mutations of CALR. We retrospectively analysed 172 ET patients diagnosed at our Institution from 1980 to 2015. In JAK2V617F- and MPLW515K/L-wild type patients CALR mutations were searched on peripheral blood and CAL2 immunostaining was performed on bone marrow. In addition, bone marrow biopsies were histologically reviewed for megakaryocytic features. Thirty-one patients (18%) were CALR-mutated. Concordance between molecular and immunohistological detection of CALR mutations was near complete, albeit a single patient was found to be positive by molecular tests only. Two patterns were defined in CAL2-positive bone marrow samples, characterized by staining of almost only megakaryocytes (pattern A: 41%) or staining of megakaryocytes and ≥ 2% small non megakaryocytic elements (pattern B: 59%), at least partially being myeloid precursors. Pattern B biopsies had higher cellularity and number of megakaryocytes compared to pattern A samples. In this series, CAL2 allowed rapid and cost-efficient identification of CALR-mutated ET patients. The biological significance of different staining pattern should be confirmed in wider and independent series.


Assuntos
Anticorpos Monoclonais/química , Especificidade de Anticorpos , Medula Óssea , Calreticulina , Mutação , Trombocitemia Essencial , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia , Medula Óssea/metabolismo , Medula Óssea/patologia , Calreticulina/genética , Calreticulina/metabolismo , Feminino , Humanos , Imuno-Histoquímica/métodos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Masculino , Megacariócitos/metabolismo , Megacariócitos/patologia , Pessoa de Meia-Idade , Estudos Retrospectivos , Trombocitemia Essencial/diagnóstico , Trombocitemia Essencial/genética , Trombocitemia Essencial/metabolismo , Trombocitemia Essencial/patologia
7.
Genomics Proteomics Bioinformatics ; 17(2): 190-200, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-31201998

RESUMO

Chimeric antigen receptor (CAR) T cell therapy has exhibited dramatic anti-tumor efficacy in clinical trials. In this study, we reported the transcriptome profiles of bone marrow cells in four B cell acute lymphoblastic leukemia (B-ALL) patients before and after CD19-specific CAR-T therapy. CD19-CAR-T therapy remarkably reduced the number of leukemia cells, and three patients achieved bone marrow remission (minimal residual disease negative). The efficacy of CD19-CAR-T therapy on B-ALL was positively correlated with the abundance of CAR and immune cell subpopulations, e.g., CD8+ T cells and natural killer (NK) cells, in the bone marrow. Additionally, CD19-CAR-T therapy mainly influenced the expression of genes linked to cell cycle and immune response pathways, including the NK cell mediated cytotoxicity and NOD-like receptor signaling pathways. The regulatory network analyses revealed that microRNAs (e.g., miR-148a-3p and miR-375), acting as oncogenes or tumor suppressors, could regulate the crosstalk between the genes encoding transcription factors (TFs; e.g., JUN and FOS) and histones (e.g., HIST1H4A and HIST2H4A) involved in CD19-CAR-T therapy. Furthermore, many long non-coding RNAs showed a high degree of co-expression with TFs or histones (e.g., FOS and HIST1H4B) and were associated with immune processes. These transcriptome analyses provided important clues for further understanding the gene expression and related mechanisms underlying the efficacy of CAR-T immunotherapy.


Assuntos
Antígenos CD19/metabolismo , Redes Reguladoras de Genes , Imunoterapia Adotiva , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Transcriptoma/genética , Adulto , Medula Óssea/metabolismo , Linfócitos T CD8-Positivos/imunologia , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Receptores de Antígenos de Linfócitos T , Fatores de Transcrição/metabolismo
8.
Cell Prolif ; 52(4): e12587, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31206838

RESUMO

OBJECTIVES: Cellular aggregates are readily applicable in cell-based therapy. The effects of agitation and inoculation density on the aggregation of cells in spinner flask and the molecular mechanism of aggregation were investigated. MATERIALS AND METHODS: The aggregation kinetics of cells in spinner flask was evaluated with bovine articular chondrocytes (bACs), rabbit bone marrow-derived mesenchymal stem cells (rMSCs) and their mixture. The morphology of cellular aggregates was studied with scanning electron microscopy and gene expression of cell adhesion-related molecules was analysed. RESULTS: It was shown that suspension culture in spinner flask induced the aggregation of bACs and rMSCs. Both cells exhibited increased aggregation rate and aggregate size with decreasing agitation rate and increasing cell inoculation density. Additionally, aggregate size increased with extended culture time. By analysing gene expression of integrin ß1 and cadherin, it was indicated that these molecules were potentially involved in the aggregation process of bACs and rMSCs, respectively. Aggregates composed of both bACs and rMSCs were also prepared, showing rMSCs in the core and bACs in the periphery. CONCLUSIONS: Cellular aggregates were prepared in dynamic suspension culture using spinner flask, the key parameters to the aggregation process were identified, and the molecular mechanism of aggregation was revealed. This would lay a solid foundation for the large-scale production of cellular aggregates for cell-based therapy, such as cartilage regeneration.


Assuntos
Condrócitos/citologia , Células-Tronco Mesenquimais/citologia , Animais , Medula Óssea/metabolismo , Medula Óssea/fisiologia , Bovinos , Adesão Celular/fisiologia , Moléculas de Adesão Celular/metabolismo , Contagem de Células/métodos , Técnicas de Cultura de Células/métodos , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Condrócitos/metabolismo , Expressão Gênica/fisiologia , Células-Tronco Mesenquimais/metabolismo , Coelhos
9.
Folia Biol (Praha) ; 65(1): 11-23, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31171078

RESUMO

Acute myeloid leukaemia (AML) is the leading form of fatal acute leukaemia in adults. AML is a heterogeneous disease with respect to responsible mutations and chromosomal abnormalities as well as to their clinicopathological image. In recent years, great progress has been made in techniques allowing detection of genetic changes in both de novo AML and in secondary AML induced by other haematological disorders or therapy, and in detection of residual disease after therapy. Accumulated knowledge allowed better understanding of the molecules and mechanisms involved not only in the formation and expansion of a primary leukaemia-founding clone, but also of a temporal order of changes leading to the fully malignant phenotype. The recent knowledge of bone marrow (BM) compartments and interrelations among various BM resident and recruited cell types helps in understanding the AML development. The progress in the techniques and knowledge will result in the development and use of molecularly targeted therapies tailored to individual patient needs.


Assuntos
Leucemia Mieloide Aguda/metabolismo , Medula Óssea/metabolismo , Aberrações Cromossômicas , Humanos , Leucemia Mieloide Aguda/genética , Mutação/genética , Fenótipo
10.
Medicine (Baltimore) ; 98(23): e15948, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31169718

RESUMO

CXC chemokine receptor 4 (CXCR4) expression on acute myeloid leukemia (AML) cells correlated with stromal cell derived factor-1α (SDF-1α) and retained hematopoietic progenitors and leukemia cells within the bone marrow microenvironment. Here, we examined CXCR4 expression in 134 de novo AML and 21 controls by flow cytometry, evaluated the relationship between CXCR4 expression and clinical characteristics, and elucidated the prognostic significance of CXCR4 expression in AML prospectively. We found that the CXCR4 expression was significantly higher in AML patients than controls (P = .000). One hundred thirty four cases of de novo AML patients were divided into 2 groups according to the median of CXCR4 relative fluorescence intensity (RFI). CXCR4 high group (RFI >4.23) had markedly shorter overall survival (OS) and disease-free survival (DFS) than CXCR4 low group (RFI ≤4.23) in 106 AML patients who received chemotherapy (P = .002; .026, respectively). Furthermore, in the 87 non-M3 patients who received induction therapy, there was a significant decrease for OS but not for DFS in the CXCR4 high group (P = .047 and .178, respectively). Moreover, high levels of CXCR4 expression independently increased the risk of relapse in both all AML and non-M3 patients who achieved complete remission (CR) after chemotherapy (odds ratio = 1.090, P = .010; odds ratio = 1.068, P = .048, respectively). Collectively, our data suggest that CXCR4 overexpression was an independent prognostic factor for disease relapse and poorer OS in both all AML and non-M3 patients. CXCR4 expression levels can be determined at disease presentation by the flow rapidly and easily. As such, CXCR4 could be used as a potential therapeutic target in AML patients with poor prognosis.


Assuntos
Leucemia Mieloide Aguda/mortalidade , Receptores CXCR4/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Medula Óssea/metabolismo , Intervalo Livre de Doença , Feminino , Citometria de Fluxo , Humanos , Leucemia Mieloide Aguda/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Recidiva , Microambiente Tumoral , Adulto Jovem
11.
Mol Med Rep ; 19(6): 4743-4752, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059030

RESUMO

Interleukin 17A (IL­17A) exerts pleiotropic effects on periodontitis, partially through enhancement of alveolar bone loss. Osteoclasts are the main culprits that absorb alveolar bone. However, studies describing the correlation between IL­17A and osteoclasts are not conclusive. Previously, autophagy was revealed to be involved in osteoclast differentiation and bone resorption. However, the role of autophagy in IL­17A­mediated osteoclast formation is yet to be clarified. In the present study, bone marrow macrophages (BMMs) were treated with or without IL­17A. 3­Methyladenine (3­MA) was applied to inhibit autophagy. Osteoclast formation was detected by tartrate­resistant acid phosphatase (TRAP) staining, immunofluorescence, and scanning electron microscope. The effects of IL­17A on osteoclast­specific genes and autophagy­related genes during osteoclast differentiation were examined by real­time quantitative polymerase chain reaction and western blot analysis. Autophagosomes were observed by transmission electron microscope. Hematoxylin and eosin (H&E), and TRAP staining was adopted to assess alveolar bone destruction and the number of osteoclasts, respectively in a rat periodontitis model. Consequently, IL­17A stimulated osteoclast differentiation and bone resorption of BMMs accompanied by an increase in the mRNA expression of osteoclast­specific genes. Furthermore, IL­17A increased the levels of autophagy­related genes and proteins, and inhibition of autophagy with 3­MA attenuated the IL­17A­mediated osteoclastogenesis. In addition, there was an increase in the number of osteoclasts and alveolar bone resorption with IL­17A treatment in the periodontitis rat model. Collectively, these findings indicated that IL­17A facilitated osteoclast differentiation and bone resorption in vitro and in vivo, which may contribute to the understanding of the molecular basis of IL­17A in alveolar bone destruction and provide insight on the clinical therapeutic targets for periodontitis.


Assuntos
Autofagia/efeitos dos fármacos , Medula Óssea/metabolismo , Reabsorção Óssea/metabolismo , Diferenciação Celular/efeitos dos fármacos , Interleucina-17/farmacologia , Macrófagos/metabolismo , Osteoclastos/metabolismo , Actinas/metabolismo , Adenina/análogos & derivados , Adenina/antagonistas & inibidores , Perda do Osso Alveolar/tratamento farmacológico , Perda do Osso Alveolar/patologia , Animais , Autofagossomos/patologia , Autofagia/genética , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Reabsorção Óssea/patologia , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/genética , Periodontite/tratamento farmacológico , Periodontite/patologia , Ratos , Ratos Sprague-Dawley
12.
Cancer Sci ; 110(8): 2421-2430, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31145521

RESUMO

Although the targeted tyrosine kinase inhibitor imatinib mesylate (IM) has achieved significant responses against CML in the clinical setting, a small proportion of patients fail to respond to IM treatment and their disease continues to progress, indicating resistance to IM therapy. As a secreted extracellular matrix protein, cysteine-rich protein 61 (Cyr61) plays an important role in the resistance of solid tumors to chemotherapy, but its role in CML is unclear. In the present study, we observed that Cyr61 levels were upregulated in the plasma and bone marrow (BM) of patients with CML as well as in K562 cells. This upregulation of Cyr61 significantly decreased IM-induced cellular apoptosis of K562 cells through nuclear factor kappa B/B-cell lymphoma 2 pathways. Inhibition of Cyr61 restored the chemosensitivity of K562 cells to IM both in vitro and in vivo. Thus, our results showed for the first time that Cyr61 plays an important role in regulating the chemosensitivity of CML cells to IM, suggesting that selectively targeting Cyr61 directly or its relevant effector pathways may provide potential value in improving the clinical response of patients with CML to IM treatment.


Assuntos
Proteína Rica em Cisteína 61/metabolismo , Mesilato de Imatinib/farmacologia , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Apoptose/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Inibidores de Proteínas Quinases/farmacologia , Regulação para Cima/efeitos dos fármacos
13.
Int J Mol Sci ; 20(10)2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-31100865

RESUMO

Adult metabolic syndrome is considered to be elicited by the developmental programming which is regulated by the prenatal environment. The maternal excess intake of fructose, a wildly used food additive, is found to be associated with developmental programing-associated cardiovascular diseases. To investigate the effect of maternal fructose exposure (MFE) on endothelial function and repair, which participate in the initiation and progress of cardiovascular disease, we applied a rat model with maternal fructose excess intake during gestational and lactational stage and examined the number and function of endothelial progenitor cells (EPCs) in 3-month-old male offspring with induction of critical limb ischemia (CLI). Results showed that the circulating levels of c-Kit+/CD31+ and Sca-1+/KDR+ EPC were reduced by MFE. In vitro angiogenesis analysis indicated the angiogenic activity of bone marrow-derived EPC, including tube formation and cellular migration, was reduced by MFE. Western blots further indicated the phosphorylated levels of ERK1/2, p38-MAPK, and JNK in circulating peripheral blood mononuclear cells were up-regulated by MFE. Fourteen days after CLI, the reduced blood flow recovery, lowered capillary density, and increased fibrotic area in quadriceps were observed in offspring with MFE. Moreover, the aortic endothelium-mediated vasorelaxant response in offspring was impaired by MFE. In conclusion, maternal fructose intake during gestational and lactational stage modulates the number and angiogenic activity of EPCs and results in poor blood flow recovery after ischemic injury.


Assuntos
Células Progenitoras Endoteliais/metabolismo , Frutose/metabolismo , Frutose/farmacologia , Isquemia/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Fluxo Sanguíneo Regional , Animais , Ataxina-1 , Medula Óssea/metabolismo , Doenças Cardiovasculares , Movimento Celular , Modelos Animais de Doenças , Extremidades/patologia , Isquemia/patologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Sistema de Sinalização das MAP Quinases , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas , Proteínas Proto-Oncogênicas c-kit , Ratos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular
14.
Ann Hematol ; 98(8): 1905-1918, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31104089

RESUMO

Efficient and safe delivery of siRNA in vivo is the biggest roadblock to clinical translation of RNA interference (RNAi)-based therapeutics. To date, lipid nanoparticles (LNPs) have shown efficient delivery of siRNA to the liver; however, delivery to other organs, especially hematopoietic tissues still remains a challenge. We developed DLin-MC3-DMA lipid-based LNP-siRNA formulations for systemic delivery against a driver oncogene to target human chronic myeloid leukemia (CML) cells in vivo. A microfluidic mixing technology was used to obtain reproducible ionizable cationic LNPs loaded with siRNA molecules targeting the BCR-ABL fusion oncogene found in CML. We show a highly efficient and non-toxic delivery of siRNA in vitro and in vivo with nearly 100% uptake of LNP-siRNA formulations in bone marrow of a leukemic model. By targeting the BCR-ABL fusion oncogene, we show a reduction of leukemic burden in our myeloid leukemia mouse model and demonstrate reduced disease burden in mice treated with LNP-BCR-ABL siRNA as compared with LNP-CTRL siRNA. Our study provides proof-of-principle that fusion oncogene specific RNAi therapeutics can be exploited against leukemic cells and promise novel treatment options for leukemia patients.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Nanopartículas/administração & dosagem , RNA Interferente Pequeno/farmacologia , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/patologia , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Expressão Gênica , Marcação de Genes/métodos , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Lipídeos/administração & dosagem , Lipídeos/química , Camundongos , Camundongos Nus , Nanopartículas/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacocinética , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Ann Hematol ; 98(7): 1713-1720, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31053880

RESUMO

Symptomatic multiple myeloma (MM) is a plasma cell neoplasm that represents the final stage of a continuum of clinical conditions that start from monoclonal gammopathy of unknown significance (MGUS), then transits in the more advance, but still asymptomatic, smoldering MM (SMM), with a final evolution in symptomatic MM. To investigate SMM microenvironment modifications, we studied 16 patients diagnosed at our hospital. Eight of them (group A) developed MM within 2 years from diagnosis while the others (group B) had stable SMM. Samples were bone marrow biopsies at diagnosis and after 2 years (± 4 months) and were analyzed by immunohistochemical analysis. Firstly, we found a significant increase in both CD4+ cells (11 vs 17%, p < 0.01) and CD8+ cells (15 vs 18%, p < 0.01) between diagnosis and at follow-up samples (whole cohort). This was associated to an increase in the CD4+/CD8+ ratio (0.74 vs 0.93, p < 0.01). Secondly, we discovered an increased expression of T cell inhibitory molecules during SMM evolution. In fact, plasma cell PD-L1 and microenvironment cell LAG3 expression increased from 1 to 12% (p = 0.03) and 4 to 10% (p = 0.04), respectively, from diagnosis to follow-up. Also, plasma cells and microenvironment cells HLA-DR expression augmented during SMM evolution from 7 to 10% (p = 0.04) and 29 to 39% (p = 0.01), respectively. When comparing group A vs group B, we found an increased CD68-KP1+ cell infiltration in favor of group B at diagnosis (23 vs 28%, p = 0.01) and a greater plasma cell infiltration at follow-up (50 vs 26%, p < 0.01). Our findings suggest how immune escape mechanisms appear earlier during multiple myeloma evolution, and that LAG3 could be a possible immunologic target in this setting.


Assuntos
Antígenos CD/biossíntese , Antígeno B7-H1/biossíntese , Regulação Neoplásica da Expressão Gênica , Antígenos HLA-DR/biossíntese , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/biossíntese , Mieloma Múltiplo Latente , Biópsia , Medula Óssea/metabolismo , Medula Óssea/patologia , Relação CD4-CD8 , Feminino , Humanos , Masculino , Mieloma Múltiplo/patologia , Plasmócitos/metabolismo , Plasmócitos/patologia , Estudos Retrospectivos , Mieloma Múltiplo Latente/metabolismo , Mieloma Múltiplo Latente/patologia , Evasão Tumoral , Microambiente Tumoral
16.
Vet Immunol Immunopathol ; 211: 19-24, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31084889

RESUMO

Porcine reproductive and respiratory syndrome (PRRS) is one of the most common diseases in the global swine industry. PRRSV infection is highly restricted to cells of the monocyte-macrophage lineage. However, the lack of antibodies to swine monocyte-macrophage lineage markers significantly hampers PRRSV research. In this study, we have developed a monoclonal antibody against the swine leukocyte antigen (SLA)-DRα chain and confirmed its reactivity with endogenous expressed SLA-DR in a variety of cell lines and primary swine antigen-presenting cells (PAMs, PBMC and BM-DCs). Moreover, the level of SLA-DR expression after PRRSV infection were evaluated by our homemade Mab and a commercial anti-SLA-DR antibody. Based on our result, the protein level of SLA-DRα expression is increased after PRRSV infection in DC, while the mRNA of both SLA-DRα and SLA-DRß were significantly inhibited by PRRSV replication. In conclusion, we successfully developed a MAb reactive with endogenous SLA-DR in western blotting, and this MAb could be a useful tool for further research and analysis. Moreover, the inconsistency of SLA-DR expression between protein and mRNA levels may suggest a novel role of DC played during the immune response after PRRSV infection.


Assuntos
Anticorpos Monoclonais/imunologia , Células Dendríticas/metabolismo , Cadeias alfa de HLA-DR/imunologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Animais , Western Blotting , Medula Óssea/imunologia , Medula Óssea/metabolismo , Linhagem Celular , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Células HEK293 , Cadeias alfa de HLA-DR/metabolismo , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos BALB C/imunologia , Proteínas Recombinantes , Suínos/imunologia
17.
Biomed Pharmacother ; 114: 108806, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30928804

RESUMO

Agents that provide protection against irradiation-induced hematopoietic injury are urgently needed for radiotherapy. We examined the effects of the small molecule, 1,2-propanediol (PPD), on total body irradiation (TBI)-induced hematopoietic injury in C57BL/6 mice. PPD administration 1 h before TBI significantly increased hematopoietic parameters such as white blood cell, platelet, red blood cell, and lymphocyte counts in vivo and enhanced the survival of mice exposed to TBI (7.0 and 7.5 Gy). PPD administration 1 h before TBI improved bone marrow (BM) and spleen recovery after TBI, with increases in both BM cellularity and spleen index. The number of colony-forming-units in bone marrow mononuclear cells (BMNCs) in vitro also increased significantly. PPD pretreatment increased the numbers of hematopoietic stem cells and hematopoietic progenitor cells in BM. Importantly, PPD also maintained endogenous antioxidant status by decreasing levels of malondialdehyde and increasing the expression of reduced glutathione, superoxide dismutase and catalase in the serum of irradiated mice. PPD alleviated the levels of apoptosis in HSCs induced by TBI, thus increasing the proportion of dividing BMNCs. These results suggest that PPD protects against TBI-induced hematopoietic injury through the increased activities of antioxidant enzymes and the inhibition of apoptosis in HSCs. PPD increased the serum levels of granulocyte-colony stimulating factor and interleukin-6 irrespective of TBI. In conclusion, these data suggest that PPD acts as a radioprotector against radiation-induced hematopoietic injury.


Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Propilenoglicol/farmacologia , Lesões Experimentais por Radiação/tratamento farmacológico , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Ensaio de Unidades Formadoras de Colônias/métodos , Fator Estimulador de Colônias de Granulócitos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Lesões Experimentais por Radiação/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Irradiação Corporal Total/métodos
18.
Int J Mol Sci ; 20(8)2019 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-31013941

RESUMO

Myeloproliferative neoplasms represent a heterogenous group of disorders of the hematopoietic stem cell, with an intrinsic risk of evolution into acute myeloid leukemia. The frequency of leukemic evolution varies according to myeloproliferative neoplasms subtype. It is highest in primary myelofibrosis, where it is estimated to be approximately 10-20% at 10 years, following by polycythemia vera, with a risk of 2.3% at 10 years and 7.9% at 20 years. In essential thrombocythemia, however, transformation to acute myeloid leukemia is considered relatively uncommon. Different factors are associated with leukemic evolution in myeloproliferative neoplasms, but generally include advanced age, leukocytosis, exposure to myelosuppressive therapy, cytogenetic abnormalities, as well as increased number of mutations in genes associated with myeloid neoplasms. The prognosis of these patients is dismal, with a medium overall survival ranging from 2.6-7.0 months. Currently, there is no standard of care for managing the blast phase of these diseases, and no treatment to date has consistently led to prolonged survival and/or hematological remission apart from an allogeneic stem cell transplant. Nevertheless, new targeted agents are currently under development. In this review, we present the current evidence regarding risk factors, molecular characterization, and treatment options for this critical subset of myeloproliferative neoplasms patients.


Assuntos
Transformação Celular Neoplásica , Transtornos Mieloproliferativos/etiologia , Transtornos Mieloproliferativos/metabolismo , Animais , Biomarcadores , Crise Blástica/genética , Crise Blástica/metabolismo , Crise Blástica/patologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Aberrações Cromossômicas , Terapia Combinada , Progressão da Doença , Humanos , Leucemia Mieloide Aguda/etiologia , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Terapia de Alvo Molecular , Mutação , Transtornos Mieloproliferativos/tratamento farmacológico , Transtornos Mieloproliferativos/patologia , Fatores de Risco
19.
Acta Haematol ; 141(3): 189-198, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30840964

RESUMO

Calorie restriction (CR) has been studied as a way to prolong longevity, and CR before chemotherapy can reduce hematological toxicity in cancer patients. We investigated the influence of fasting on immune cells and immature hematopoietic cells. In fasted mice, there was a significant reduction in the hematopoietic stem cell count but no significant difference for progenitor cells. Colony assays showed no difference and the rates of early and late apoptosis were almost identical when comparing fasted and control mice. DNA cell cycle analysis of immature bone marrow (BM) cells showed that CR caused a significant increase in the percentage in the G0/G1 phase and decreases in the S and G2/M phases. We detected a remarkable increase of T cells in the BM of fasted mice. CD44- naïve CD8+ T cells were more numerous in fasted BM, as were naïve CD4+ T cells, and part of those T cells showed less tendency in the G0/G1 phase. Immature hematopoietic cells remained in a relatively quiescent state and retention of colony-forming capacity during CR. The number of naïve T cells in the BM of fasted mice increased. These findings imply immature hematopoietic cells and some lymphoid cells can survive starvation, whilst maintaining their function.


Assuntos
Medula Óssea/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Pontos de Checagem do Ciclo Celular/fisiologia , Jejum/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Células-Tronco Hematopoéticas/citologia , Camundongos
20.
Res Vet Sci ; 124: 212-222, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30925336

RESUMO

Little information is currently available on therapeutic features of bovine mesenchymal stem cells (MSCs), despite the development of large animal experimental models including cattle may open alternative strategies for investigating MSC physiology and eventual applications for regenerative therapy. The aim of the present study was to compare in vitro immunomodulatory and immunogenic potentials of bovine fetal MSCs (bfMSCs) derived from bovine fetal bone marrow (BM-MSCs) and adipose tissue (AT-MSCs). Immunomodulatory analyses in bfMSCs were performed by determination of the effect of interferon-γ (IFNγ) on mRNA levels of indoleamine 2, 3-dioxygenase (IDO), transforming growth factor ß1 (TGFß1), prostaglandin E receptor 2 (PTGER2), interleukin-6 and -10 (IL-6 and IL-10), and IDO enzymatic activity. The effect of conditioned medium from IFNγ-stimulated bfMSCs on the proliferation of alloantigen-activated peripheral blood lymphocytes (PBLs) was assessed. Immunogenicity of bfMSCs was determined by quantification of mRNA levels of major histocompatibility complex I and II (MHC-I and -II), CD80 and CD86, and the proportion of cells positive for MHC-I and -II by flowcytometry (FACS) analyses. IFNγ treatment increased IL-6, PTGER2 and IDO gene expression and activity in bfMSCs but did not affect suppressive effect on proliferation of PBLs. Lower proportion of AT-MSCs expressed MHC-I and MHC-II in comparison to BM-MSCs. In conclusion, BM-MSCs and AT-MSCs upregulated expression of immunomodulatory genes in a similar way after IFNγ stimuli. BM-MSCs and AT-MSCs in basal condition and treated with IFNγ displayed similar in vitro immunomodulatory ability. Lower expression of MHC-I and MHC-II suggest that AT-MSCs might be less immunogenic compared to BM-MSCs.


Assuntos
Tecido Adiposo/metabolismo , Células da Medula Óssea/metabolismo , Imunomodulação , Células-Tronco Mesenquimais/imunologia , Animais , Medula Óssea/metabolismo , Bovinos , Feto
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