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1.
Rinsho Ketsueki ; 60(9): 1046-1055, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31597826

RESUMO

Human iPS cells are somatic cells reprogrammed to the pluripotent state. Because of their pluripotent nature, iPS cells are now commonly used to model several developmental processes including hematopoiesis in vitro. The in vitro models can be used to study the mechanisms regulating not only normal hematopoiesis but also hematological diseases ranging from monogenic congenital disorders to genetically multifactorial malignancies. Those disease models can also be used to investigate novel treatments through procedures including high throughput drug screening. The possible clinical applications of iPS cell-derived hematopoietic cells include immunotherapy with T lymphocytes, NK cells and macrophages, and transfusion therapy with platelets and red blood cells. Platelets have now been produced from iPS cells in quantities sufficient for clinical use. By developing expandable immortalized megakaryocyte cell lines (imMKCLs), several novel drugs and turbulence-incorporated bioreactors, efficient and scalable generation of platelets was achieved. This review summarizes the current status of iPS cell research in hematopoiesis with details on iPS cell-derived platelets.


Assuntos
Plaquetas/citologia , Terapia Baseada em Transplante de Células e Tecidos , Células-Tronco Pluripotentes Induzidas/citologia , Diferenciação Celular , Eritrócitos , Hematopoese , Humanos , Imunoterapia , Células Matadoras Naturais , Macrófagos , Megacariócitos , Linfócitos T
2.
Rinsho Ketsueki ; 60(9): 1063-1069, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31597828

RESUMO

In modern hematology, research on hematopoiesis and blood cells in vertebrates, such as birds, reptiles, amphibians, and fish, is lagging. This is because there are many experimental constraints when selecting subjects other than humans and mice as research subjects. Currently, the availability of flow cytometry to count classified nucleated blood cells and utilization of whole genome information have led to novel findings. For example, in case of amphibian hematopoiesis studies, megakaryocytes have been found to be present in African clawed frogs (Xenopus laevis), which do not have platelets but have circulating nucleated thrombocytes. Moreover, we shed light on several mysteries, such as the C-terminal region in human TPO molecules not being found in birds, amphibians, and fish TPO molecules and the functional universalities of mutant CALR-MPL binding and EPO-EphB4 binding, in conjunction with comparative hematology.


Assuntos
Hematologia , Megacariócitos/citologia , Trombopoese , Vertebrados , Animais , Plaquetas , Genoma , Histologia Comparada , Humanos , Camundongos
3.
Rinsho Ketsueki ; 60(9): 1166-1175, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31597840

RESUMO

The classical myeloproliferative neoplasms (MPN), polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF), are characterized by clonal myeloproliferation without features of myelodysplasia. The diagnostic approach proposed by the World Health Organization (WHO) uses clinical features, peripheral blood counts and smear analysis, bone marrow (BM) morphology, karyotype and molecular genetic tests to classify MPN subtypes. The detection of characteristic driver mutations like JAK2V617F, JAK2 exon 12, MPL, and calrecticulin (CALR) is a major diagnostic feature. JAK2 mutations are detected in more than 90% of patients with PV and are therefore used as highly sensitive clonal marker in this subtype. However, JAK2 mutations may also occur in ET and PMF, while CALR is virtually not seen in PV. Therefore, BM remains the central diagnostic platform and is essential for distinguishing ET from pre-fibrotic PMF and diagnosing cases which do not express JAK2, MPL or CALR ('wild-type' or 'triple-negative' MPN). The standardization of relevant BM features is mandatory to recognize characteristic and easy to assess patterns that enable an accurate discrimination between the MPN subtypes. Key parameters include cellularity, erythropoiesis and neutrophil granulopoiesis in context with specific features of megakaryocytes as well as the BM fiber content, especially in early stage MPN that present with thrombocytosis and clinically mimic essential thrombocythemia.


Assuntos
Transtornos Mieloproliferativos/diagnóstico , Medula Óssea/patologia , Calreticulina/genética , Diagnóstico Diferencial , Eritropoese , Humanos , Janus Quinase 2/genética , Megacariócitos/citologia , Mutação , Neutrófilos/citologia , Policitemia Vera , Trombocitemia Essencial , Organização Mundial da Saúde
4.
Rinsho Ketsueki ; 60(7): 834-842, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31391374

RESUMO

Bone marrow (BM), the tissue specializing in the production of hematopoietic cells, consists of multiple components (e.g., extracellular matrixes, vasculatures, and stromal cells) that generate a complex three-dimensional network and several localized microenvironment. These microenvironments regulate hematopoietic stem and progenitor cells, including megakaryocyte lineage cells. In this review, we first provide an overview of the microenvironment for hematopoietic stem cells as an introduction to bone marrow microenvironment and subsequently summarize the microenvironment for megakaryocyte differentiation and maturation (megakaryopoiesis). In the last portion, we describe megakaryocyte regulation by podoplanin-positive peri-arteriolar stromal cells in the mouse bone marrow.


Assuntos
Células da Medula Óssea/citologia , Medula Óssea , Lectinas Tipo C/fisiologia , Megacariócitos/citologia , Glicoproteínas de Membrana/fisiologia , Animais , Camundongos , Trombopoese
5.
Drugs ; 79(12): 1305-1319, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31292909

RESUMO

Eltrombopag is an orally available thrombopoietin receptor agonist indicated for the treatment of immune thrombocytopenia (ITP). Beyond the effect on megakaryopoiesis, the drug also showed a stimulating effect on the hematopoietic stem cell with consistent clinical efficacy in aplastic anemia (AA) and myelodysplastic syndromes (MDS). Eltrombopag is highly effective in ITP and less so in AA and MDS. This observation underlines the importance of residual normal hematopoiesis, which is maximal in ITP, minimal/absent in AA, and dysregulated in MDS. In ITP, the drug at 50-75 mg daily induced up to 85% responses both in clinical trials and real-life studies, with the possibility of tapering and discontinuation. In AA, eltrombopag at 150 mg daily was effective in about 40% of cases relapsed/refractory to standard immunosuppression or ineligible for bone marrow transplant. In MDS, the drug seems less effective, with responses in about a quarter of patients at various schedules. The efficacy of eltrombopag in ITP, AA, and MDS suggests the existence of common immune-pathological mechanisms in these diseases, including autoimmunity against peripheral blood cells and bone marrow precursors, as well as a possible evolution of one condition into the other. Additional mechanisms of action emerging from the clinical use of eltrombopag include modulation of T-regulatory cells, restoration of Fc-γ receptor balance in phagocytes, and an iron-mobilizing effect. In this review, we analyzed the most recent literature on eltrombopag use and efficacy in patients with ITP, AA, and MDS, exploring the basis for different dosing, combined treatments, and discontinuation in each context.


Assuntos
Anemia Aplástica/tratamento farmacológico , Benzoatos/uso terapêutico , Hidrazinas/uso terapêutico , Megacariócitos/imunologia , Síndromes Mielodisplásicas/tratamento farmacológico , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , Pirazóis/uso terapêutico , Benzoatos/administração & dosagem , Benzoatos/efeitos adversos , Humanos , Hidrazinas/administração & dosagem , Hidrazinas/efeitos adversos , Imunomodulação , Imunossupressão , Púrpura Trombocitopênica Idiopática/imunologia , Pirazóis/administração & dosagem , Pirazóis/efeitos adversos , Receptores de Trombopoetina/agonistas
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.
Clin Cancer Res ; 25(16): 4898-4906, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31061068

RESUMO

PURPOSE: Myelofibrosis is characterized by bone marrow fibrosis, atypical megakaryocytes, splenomegaly, constitutional symptoms, thrombotic and hemorrhagic complications, and a risk of evolution to acute leukemia. The JAK kinase inhibitor ruxolitinib provides therapeutic benefit, but the effects are limited. The purpose of this study was to determine whether targeting AURKA, which has been shown to increase maturation of atypical megakaryocytes, has potential benefit for patients with myelofibrosis. PATIENTS AND METHODS: Twenty-four patients with myelofibrosis were enrolled in a phase I study at three centers. The objective of the study was to evaluate the safety and preliminary efficacy of alisertib. Correlative studies involved assessment of the effect of alisertib on the megakaryocyte lineage, allele burden, and fibrosis. RESULTS: In addition to being well tolerated, alisertib reduced splenomegaly and symptom burden in 29% and 32% of patients, respectively, despite not consistently reducing the degree of inflammatory cytokines. Moreover, alisertib normalized megakaryocytes and reduced fibrosis in 5 of 7 patients for whom sequential marrows were available. Alisertib also decreased the mutant allele burden in a subset of patients. CONCLUSIONS: Given the limitations of ruxolitinib, novel therapies are needed for myelofibrosis. In this study, alisertib provided clinical benefit and exhibited the expected on-target effect on the megakaryocyte lineage, resulting in normalization of these cells and reduced fibrosis in the majority of patients for which sequential marrows were available. Thus, AURKA inhibition should be further developed as a therapeutic option in myelofibrosis.See related commentary by Piszczatowski and Steidl, p. 4868.


Assuntos
Mielofibrose Primária , Aurora Quinase A , Fibrose , Humanos , Janus Quinase 2 , Megacariócitos
8.
Microbiol Immunol ; 63(6): 229-237, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31041998

RESUMO

Pseudomonas aeruginosa is a major cause of nosocomial infections and contributes to higher mortality in hospitalized individuals. Infection by P. aeruginosa triggers host immune response through activation of pathogen recognition receptors, which are present in innate cells. Several studies have reported the mechanism of P. aeruginosa induced innate immunity in multiple cell types. But so far there is no reports on response of megakaryocytes to P. aeruginosa infection. Hence, our aim was to investigate the precise role and signaling mechanism of megakaryocytes during P. aeruginosa infection. In this study, we used Mo7e cells as representatives of human megakaryocyte and found that P. aeruginosa infection induces cytotoxicity in these cells. We further demonstrated that P. aeruginosa infection modulates p38 and extracellular signal regulated kinase pathways in Mo7e cells. Protein expression profiling in P. aeruginosa lipopolysaccharide-treated Mo7e cells revealed upregulation of importin subunit ß and downregulation of metabolic enzymes. Our results suggest that P. aeruginosa infection regulates mitogen-activated protein kinases signaling pathway and importin in Mo7e cells and that this is a potential mechanism for nuclear translocation of nuclear factor binding near the κ light-chain gene in B cells and c-Jun N-terminal kinases to induce cell cytotoxicity.


Assuntos
Megacariócitos/imunologia , Megacariócitos/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/patogenicidade , Transdução de Sinais , Linhagem Celular , Testes Imunológicos de Citotoxicidade , Regulação para Baixo , Perfilação da Expressão Gênica , Humanos , Imunidade Inata , Proteínas Quinases JNK Ativadas por Mitógeno , Lipopolissacarídeos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
9.
Harefuah ; 158(3): 196-199, 2019 Mar.
Artigo em Hebraico | MEDLINE | ID: mdl-30916510

RESUMO

INTRODUCTION: Primary immune thrombocytopenic purpura (ITP) is an acquired immune-mediated disorder characterized by isolated thrombocytopenia (platelet count less than 100X109/L), caused by IgG autoantibodies which bind to platelets and megakaryocyte, T cell-mediated platelet destruction and impaired megakaryocytic function. Symptoms can manifest as petechiae, purpura, mucosal bleeding and rarely fatal intracranial hemorrhage, as well as reduced quality of life. A wide range of bleeding manifestations exists and it is impossible to tell who will bleed, when and where. The goal of treatment is to prevent severe/life-threatening bleeding. Treatment modalities target various aspects of ITP pathophysiology such as the inhibition of autoantibody production (decreased autoimmune process), modulation of T cell activity (with prolongation of platelets survival), and stimulation of platelet production. The American Society of Hematology and the International Society of Thrombosis and Hemostasis published guidelines on the treatment of ITP patients, where first line treatment focuses on inhibition of autoantibody production and platelet degradation, second-line treatments include immunosuppressive drugs and splenectomy, and third-line treatments aim to stimulate platelet production by megakaryocytes. New available strategies might change the order of treatment lines. As in other situations, treatment should be tailored according to the patient's age, life style, comorbidities and compliance.


Assuntos
Púrpura Trombocitopênica Idiopática , Adulto , Plaquetas , Humanos , Megacariócitos , Púrpura Trombocitopênica Idiopática/complicações , Púrpura Trombocitopênica Idiopática/terapia , Qualidade de Vida , Trombopoese
10.
Braz Dent J ; 30(1): 12-21, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30864641

RESUMO

This study aimed to assess the effects of low molecular weight heparin (LMWH) on alveolar bone loss (ABL), blood count, and counting of megakaryocytes and adipocytes in male Wistar rats. Forty male 60-day Wistar rats were randomly divided into four groups: Control (C), Periodontal Disease (PD), Heparin (Hp) and Heparin + Periodontal Disease (Hp+PD). LMWH was applied for 60 days at doses of 1 ml/kg/day. Blood samples were collected at baseline, 30 and 60. On day-49, PD and Hp+PD groups were subjected to ligature-induced periodontitis around second upper right molar. The left side was assessed as spontaneous alveolar bone loss. Mean ABL in the side with ligature showed significantly different between C (0.35±0.07 mm) and Hp+DP (0.49±0.09 mm) groups (p<0.001), between PD (0.55±0.11 mm) and Hp (0.32±0.06 mm) groups (p<0.001) and between Hp and Hp+DP groups (p<0.001). No significant differences were found among groups for ABL in the side without ligature. Animal weight, food intake, and water consumption showed no statistically significant difference among groups. Megakaryocytes and adipocytes were counted using optical microscopy and no statistically significant differences were found. Within-groups, there were an increase and a decrease, respectively, in the counting of lymphocytes (p=0.005 for C and p=0.009 for Hp+PD groups only) and leukocytes (p=0.003 for C, p=0.001 for PD, p=0.002 for Hp, and p<0.001 for Hp+PD groups). There was no decrease in the number of platelets in the three collection periods. LMWH was not able to affect ABL, but it may change the blood counting, especially increasing lymphocytes.


Assuntos
Perda do Osso Alveolar/prevenção & controle , Heparina de Baixo Peso Molecular/farmacologia , Adipócitos/citologia , Animais , Relação Dose-Resposta a Droga , Heparina de Baixo Peso Molecular/administração & dosagem , Masculino , Megacariócitos/citologia , Ratos , Ratos Wistar
12.
Vox Sang ; 114(4): 330-339, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30900265

RESUMO

BACKGROUND AND OBJECTIVES: Several sources of haematopoietic stem cells have been used for static culture of megakaryocytes to produce platelets in vitro. This study compares and characterizes platelets produced in shear flow using precursor cells from either umbilical (UCB) or adult peripheral blood (PB). MATERIALS AND METHODS: The efficiency of platelet production of the cultured cells was studied after perfusion in custom-built von Willebrand factor-coated microfluidic flow chambers. Platelet receptor expression and morphology were investigated by flow cytometry and microscopy, respectively. RESULTS: Proliferation of stem cells isolated out of UCB was significantly higher (P < 0·0001) compared to PB. Differentiation of these cells towards megakaryocytes was significantly lower from PB compared to UCB where the fraction of CD42b/CD41 double positive events was 44 ± 9% versus 76 ± 11%, respectively (P < 0·0001). However, in vitro platelet production under hydrodynamic conditions was more efficient with 7·4 platelet-like particles per input cell from PB compared to 4·2 from UCB (P = 0·02). The percentage of events positive for CD42b, CD41 and CD61 was comparable between both stem cell sources. The mean number of receptors per platelet from UCB and PB was similar to that on blood bank platelets with on average 28 000 CD42b, 57 000 CD61 and 5500 CD49b receptors. Microscopy revealed platelets appearing similar to blood bank platelets in morphology, size and actin cytoskeleton, alongside smaller fragments and source megakaryocytes. CONCLUSION: This characterization study suggests that platelets produced in vitro under flow either from UCB or from PB share receptor expression and morphology with donor platelets stored in the blood bank.


Assuntos
Plaquetas/citologia , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Dispositivos Lab-On-A-Chip , Citoesqueleto de Actina/metabolismo , Antígenos CD34/metabolismo , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Citometria de Fluxo , Humanos , Integrina beta3/metabolismo , Megacariócitos/citologia , Microscopia , Fenótipo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Refrigeração
13.
Nat Commun ; 10(1): 1270, 2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894540

RESUMO

Gfi1b is a transcriptional repressor expressed in hematopoietic stem cells (HSCs) and megakaryocytes (MKs). Gfi1b deficiency leads to expansion of both cell types and abrogates the ability of MKs to respond to integrin. Here we show that Gfi1b forms complexes with ß-catenin, its co-factors Pontin52, CHD8, TLE3 and CtBP1 and regulates Wnt/ß-catenin-dependent gene expression. In reporter assays, Gfi1b can activate TCF-dependent transcription and Wnt3a treatment enhances this activation. This requires interaction between Gfi1b and LSD1 and suggests that a tripartite ß-catenin/Gfi1b/LSD1 complex exists, which regulates Wnt/ß-catenin target genes. Consistently, numerous canonical Wnt/ß-catenin target genes, co-occupied by Gfi1b, ß-catenin and LSD1, have their expression deregulated in Gfi1b-deficient cells. When Gfi1b-deficient cells are treated with Wnt3a, their normal cellularity is restored and Gfi1b-deficient MKs regained their ability to spread on integrin substrates. This indicates that Gfi1b controls both the cellularity and functional integrity of HSCs and MKs by regulating Wnt/ß-catenin signaling pathway.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Megacariócitos/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Via de Sinalização Wnt , Proteína Wnt3A/genética , beta Catenina/genética , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Animais , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Ontologia Genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Células K562 , Megacariócitos/citologia , Camundongos , Camundongos Knockout , Anotação de Sequência Molecular , Cultura Primária de Células , Proteínas Proto-Oncogênicas/deficiência , Proteínas Repressoras/deficiência , Tamoxifeno , Proteína Wnt3A/metabolismo , beta Catenina/metabolismo
14.
Forensic Sci Int ; 297: 315-325, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30852415

RESUMO

Providing evidence of asphyxia death is a challenging issue in forensic pathology. Besides helpful macroscopical signs (e.g. strangulation mark, lung edema), recent data from literature indicate that the time of protracted asphyxia suffices to trigger an increase of giant cells and a migration of inflammatory cells from the bone marrow to the lung, thus offering a help in diagnosis of asphyxia death. In search of new valid asphyxia markers, the present study examined this hypothesis and investigated the leading role of pre-existing lung tissue cells and their functional state in reaction patterns to asphyxia. In specimens of suffocated human lungs following a short (n = 13) and a long asphyxia terminal episode (n = 15), and controls (sudden cardiovascular (n = 11) and traumatic deaths (n = 7)), the count of alveolar phagocytes, megakaryocytes, giant and mast cells, using H&E and toluidine blue staining, was performed. To show macrophage activation, immunohistochemical stainings for CD68, late (25F9) and early (MRP-8/-14) stage inflammatory markers were used. Measuring concentration of tryptase in femoral blood acted as a parameter for mast cell degranulation and consequently their activation. Results showed the lack of specificity of macroscopical parameters despite an association of suffocation with heavy lung edema. No significant differences in the numbers of inflammatory cells in the lungs of different case groups were detected. The doubling of MRP-8- and a five-fold elevation of MRP-14-positive cells compared to cardiovascular controls, proved an early activation state of pre-exiting monocytes in protracted asphyxia. These activated monocytes induced the degranulation of mast cells, resulting in slightly elevated tryptase levels in suffocation compared to cardiovascular controls. In summary, the duration of asphyxia (max. 20 min in cases investigated) only suffices to cause changes on molecular level, being not detectable in any specific macroscopical or histological form in the lung. Despite a potential utility of this molecular insight in individual cases, these results point to the classic doctrine of the evaluation of a rounded overall picture, accentuating on the proof of the ligature tool and the marks of suffocation process.


Assuntos
Asfixia/patologia , Pulmão/patologia , Alvéolos Pulmonares/patologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Biomarcadores/metabolismo , Calgranulina B/metabolismo , Estudos de Casos e Controles , Contagem de Células , Feminino , Patologia Legal , Humanos , Hipóxia/patologia , Imuno-Histoquímica , Pulmão/metabolismo , Ativação de Macrófagos , Macrófagos Alveolares , Masculino , Mastócitos/patologia , Megacariócitos/patologia , Pessoa de Meia-Idade , Monócitos/patologia , Fagócitos/patologia , Mudanças Depois da Morte , Triptases/sangue
15.
PLoS Comput Biol ; 15(3): e1006775, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30840616

RESUMO

BACKGROUND: Thrombocytopenia is a major side-effect of cytotoxic cancer therapies. The aim of precision medicine is to develop therapy modifications accounting for the individual's risk. METHODOLOGY/PRINCIPLE FINDINGS: To solve this task, we develop an individualized bio-mechanistic model of the dynamics of bone marrow thrombopoiesis, circulating platelets and therapy effects thereon. Comprehensive biological knowledge regarding cell differentiation, amplification, apoptosis rates, transition times and corresponding regulations are translated into ordinary differential equations. A model of osteoblast/osteoclast interactions was incorporated to mechanistically describe bone marrow support of quiescent cell stages. Thrombopoietin (TPO) as a major regulator is explicitly modelled including pharmacokinetics and-dynamics of TPO injections. Effects of cytotoxic drugs are modelled by transient depletions of proliferating cells. To calibrate the model, we used population data from the literature and close-meshed individual data of N = 135 high-grade non-Hodgkin's lymphoma patients treated with CHOP-like chemotherapies. To limit the number of free parameters, several parsimony assumptions were derived from biological data and tested via Likelihood methods. Heterogeneity of patients was explained by a few model parameters. The over-fitting issue of individual parameter estimation was successfully dealt with a virtual participation of each patient in population-based experiments. The model qualitatively and quantitatively explains a number of biological observations such as the role of osteoblasts in explaining long-term toxic effects, megakaryocyte-mediated feedback on stem cells, bi-phasic stimulation of thrombopoiesis by TPO, dynamics of megakaryocyte ploidies and non-exponential platelet degradation. Almost all individual time series could be described with high precision. We demonstrated how the model can be used to provide predictions regarding individual therapy adaptations. CONCLUSIONS: We propose a mechanistic thrombopoiesis model of unprecedented comprehensiveness in both, biological mechanisms considered and experimental data sets explained. Our innovative method of parameter estimation allows robust determinations of individual parameter settings facilitating the development of individual treatment adaptations during chemotherapy.


Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Modelos Biológicos , Trombocitopenia/induzido quimicamente , Trombopoese/efeitos dos fármacos , Antineoplásicos/efeitos adversos , Plaquetas/citologia , Humanos , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/patologia , Megacariócitos/citologia , Polietilenoglicóis/metabolismo , Células-Tronco/citologia , Trombopoetina/metabolismo , Fatores de Tempo
16.
Zhonghua Nei Ke Za Zhi ; 58(4): 282-287, 2019 Apr 01.
Artigo em Chinês | MEDLINE | ID: mdl-30917421

RESUMO

Objective: To explore the predictive factors for determining the therapeutic response and prognosis of severe thrombocytopenia (TP) in patients with primary Sjögren syndrome(pSS). Method: Patients with pSS and severe TP (platelet count ≤ 50×10(9)/L) admitted between 2010 to 2016 at Peking Union Medical College Hospital were classified according to their therapeutic response and analyzed retrospectively. The response parameters and clinical data including bone marrow aspiration results and laboratory findings were collected. Result: Thirty patients were finally analyzed, including twenty with appreciable bone marrow aspiration results. Fourteen and 7 patients achieved a complete response (CR) and a partial response (PR) respectively, other 9 patients with no response (NR). The megakaryocyte counts in bone marrow (BM-MK) counts per slide in each group were 13.0 (9.2,23.5) in CR patients, 7.0 (7.0,20.0) in PR patients, and 5.0 (1.0,6.0) in NR patients. BM-MK counts in patients with clinical response (CR+PR) were significantly higher than those with NR (P=0.006). A receiver-operation characteristic analysis revealed a cutoff value of BM-MK counts at 6.5 per slide stratifying patients by different responses with a sensitivity of 13/14, a specificity of 6/7, and area under the curve of 0.879. Univariate analysis indicated a better prognosis as BM-MK counts>6.5 per slide. Conclusion: BM-MK count could be a predictive factor of response in patients with pSS and severe TP. Patients with BM-MK counts≤6.5 per slide represent worse platelet improvement..


Assuntos
Megacariócitos , Síndrome de Sjogren , Medula Óssea , Humanos , Contagem de Plaquetas , Estudos Retrospectivos , Trombocitopenia
17.
Proc Natl Acad Sci U S A ; 116(11): 4983-4988, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30804189

RESUMO

Platelets mediate primary hemostasis, and recent work has emphasized platelet participation in immunity and inflammation. The function of the platelet-specific integrin αIIbß3 as a fibrinogen receptor in hemostasis is well defined, but the roles of αIIbß3 or integrin-associated proteins in nonhemostatic platelet functions are poorly understood. Here we show that human platelets express the integrin-associated protein SHARPIN with functional consequences. In leukocytes, SHARPIN interacts with integrin α cytoplasmic tails, and it is also an obligate member of the linear ubiquitin chain assembly complex (LUBAC), which mediates Met1 linear ubiquitination of proteins leading to canonical NF-κB activation. SHARPIN interacted with αIIb in pull-down and coimmunoprecipitation assays. SHARPIN was partially localized, as was αIIbß3, at platelet edges, and thrombin stimulation induced more central SHARPIN localization. SHARPIN also coimmunoprecipitated from platelets with the two other proteins comprising LUBAC, the E3 ligase HOIP and HOIL-1. Platelet stimulation with thrombin or inflammatory agonists, including lipopolysaccharide or soluble CD40 ligand (sCD40L), induced Met1 linear ubiquitination of the NF-κB pathway protein NEMO and serine-536 phosphorylation of the p65 RelA subunit of NF-κB. In human megakaryocytes and/or platelets derived from induced pluripotent stem (iPS) cells, SHARPIN knockdown caused increased basal and agonist-induced fibrinogen binding to αIIbß3 as well as reduced Met1 ubiquitination and RelA phosphorylation. Moreover, these SHARPIN knockdown cells exhibited increased surface expression of MHC class I molecules and increased release of sCD40L. These results establish that SHARPIN functions in the human megakaryocyte/platelet lineage through protein interactions at the nexus of integrin and immune/inflammatory signaling.


Assuntos
Plaquetas/metabolismo , Transdução de Sinais , Ubiquitinas/metabolismo , Linhagem da Célula , Técnicas de Silenciamento de Genes , Homeostase , Humanos , Quinase I-kappa B/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Inflamação/patologia , Megacariócitos/metabolismo , Modelos Biológicos , NF-kappa B/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Ligação Proteica , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
18.
Biochem Biophys Res Commun ; 510(3): 456-461, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30732856

RESUMO

Platelet, apart from its classic role of homeostasis, serves also as a crucial immune cell component that contributes to the aggravation of atherosclerosis. It has been reported that myocardial infarction (MI) triggers leukocytosis in the bone marrow and spleen, which accelerates post-MI atherosclerosis. However, it remains unclear whether thrombopoiesis is also enhanced after MI. Here, using flow cytometry and bone marrow whole-mount immunofluorescence staining combined with three-dimensional (3D) reconstruction, we for the first time demonstrated an enhanced thrombopoiesis and megakaryopoiesis in a mouse model of coronary artery ligation as a mimic of MI. We showed that MI leads to increasing number of peripheral platelets, as well as elevating number and larger size of bone marrow MKs. We also observed more proplatelets and fragmented MKs, and a closer spatial distribution of MK populations to the bone marrow vascular niche after MI. This study provides direct evidence that MI induces bone marrow megakaryocyte proliferation, maturation and platelet production. It opens a new scope that targeting platelet production might become a novel therapeutic approach for attenuating post-MI atherosclerosis.


Assuntos
Plaquetas/fisiologia , Megacariócitos/citologia , Infarto do Miocárdio/fisiopatologia , Trombopoese , Animais , Proliferação de Células , Masculino , Células Progenitoras de Megacariócitos/fisiologia , Megacariócitos/fisiologia , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia
20.
Mol Immunol ; 107: 123-131, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30738249

RESUMO

This study was conducted to investigate the effect of CD226 on the differentiation, activation, and polyploidization of megakaryocytes (MKs) and explore the potential mechanism. Dami (megakaryocyte line) cell maturation was induced by phorbol 12-myristate 13-acetate. CD226 was silenced by infection with a CD226-specific shRNA lentiviral vector. The mRNA level of CD226 was detected by qRT-PCR. The expressions of Dami cells surface CD226, MK specific markers CD41 and CD62P, and DNA ploidy in Dami cells and CD226 knockdown (KD) cells were evaluated by flow cytometry. The effect of CD226 on the expression of megakaryocyte-associated transcription factors was measured by western blot and confocal analysis. Transfection with CD226 shRNA lentivirus dramatically decreased the level of CD226 and expression of CD62 P in Dami cells. Silencing of CD226 caused morphological changes and differentiation retardation in low-ploidy MK. Furthermore, CD226 knockout (KO) mice exhibited increased 2N-4N low-ploidy MK and decreased ≥8N polyploidy. Interestingly, silencing of CD226 in megakaryocytic cells down-regulated the expression of early stage transcription factors includes GATA-binding factor 1 (GATA-1) and friend leukemia integration 1 (FLI-1), but not late-stage nuclear factor, erythroid 2 (NF-E2). CD226 is involved in MKs activation and polyploidy cell cycle control.


Assuntos
Antígenos de Diferenciação de Linfócitos T/imunologia , Diferenciação Celular/imunologia , Regulação da Expressão Gênica/imunologia , Megacariócitos/imunologia , Animais , Antígenos de Diferenciação de Linfócitos T/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem Celular , Fator de Transcrição GATA1/genética , Fator de Transcrição GATA1/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Megacariócitos/citologia , Camundongos , Camundongos Knockout , Subunidade p45 do Fator de Transcrição NF-E2/genética , Subunidade p45 do Fator de Transcrição NF-E2/imunologia , Selectina-P/genética , Selectina-P/imunologia , Ploidias , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/imunologia , Acetato de Tetradecanoilforbol/farmacologia
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