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1.
Stem Cell Res Ther ; 15(1): 178, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38886845

RESUMO

BACKGROUND: Bone marrow stromal cells (BMSCs) are highly heterogeneous, which may reflect their diverse biological functions, including tissue maintenance, haematopoietic support and immune control. The current understanding of the mechanisms that drive the onset and resolution of heterogeneity, and how BMSCs influence other cells in their environment is limited. Here, we determined how the secretome and importantly the extracellular matrix of BMSCs can influence cellular phenotype. METHODS: We used two immortalised clonal BMSC lines isolated from the same heterogeneous culture as model stromal subtypes with distinct phenotypic traits; a multipotent stem-cell-like stromal line (Y201) and a nullipotent non-stem cell stromal line (Y202), isolated from the same donor BMSC pool. Label-free quantitative phase imaging was used to track cell morphology and migration of the BMSC lines over 96 h in colony-forming assays. We quantified the secreted factors of each cell line by mass spectrometry and confirmed presence of proteins in human bone marrow by immunofluorescence. RESULTS: Transfer of secreted signals from a stem cell to a non-stem cell resulted in a change in morphology and enhanced migration to more closely match stem cell-like features. Mass spectrometry analysis revealed a significant enrichment of extracellular matrix (ECM) proteins in the Y201 stem cell secretome compared to Y202 stromal cells. We confirmed that Y201 produced a more robust ECM in culture compared to Y202. Growth of Y202 on ECM produced by Y201 or Y202 restored migration and fibroblastic morphology, suggesting that it is the deficiency of ECM production that contributes to its phenotype. The proteins periostin and aggrecan, were detected at 71- and 104-fold higher levels in the Y201 versus Y202 secretome and were subsequently identified by immunofluorescence at rare sites on the endosteal surfaces of mouse and human bone, underlying CD271-positive stromal cells. These proteins may represent key non-cellular components of the microenvironment for bona-fide stem cells important for cell maintenance and phenotype in vivo. CONCLUSIONS: We identified plasticity in BMSC morphology and migratory characteristics that can be modified through secreted proteins, particularly from multipotent stem cells. Overall, we demonstrate the importance of specific ECM proteins in co-ordination of cellular phenotype and highlight how non-cellular components of the BMSC microenvironment may provide insights into cell population heterogeneity and the role of BMSCs in health and disease.


Assuntos
Matriz Extracelular , Células-Tronco Mesenquimais , Fenótipo , Humanos , Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Movimento Celular , Proteínas da Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/genética , Células Estromais/metabolismo , Células Estromais/citologia , Linhagem Celular
2.
Orphanet J Rare Dis ; 19(1): 203, 2024 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-38760658

RESUMO

BACKGROUND: Activated phosphoinositide 3-kinase delta syndrome (APDS) is an ultra-rare primary immunodeficiency, with only 256 cases reported globally. This study aimed to explore the disease burden of APDS from the perspective of individuals with APDS and their caregivers. METHODS: Qualitative interviews were conducted with healthcare providers (HCPs), individuals with APDS and caregivers, to explore the symptoms and health-related quality of life (HRQoL) impact of APDS. Some individuals and caregivers also completed a narrative account exercise. All interviews were audio recorded and transcribed. Data were analysed using thematic analysis and saturation was recorded. RESULTS: Semi-structured qualitative interviews were conducted with healthcare providers (HCPs), individuals with APDS and caregivers. Individuals and caregivers had the option of completing a narrative account exercise. Six HCPs participated in an interview. Seven participants completed the narrative account exercise (N = 5 caregivers and N = 2 individuals with APDS) and 12 took part in an interview (N = 4 caregivers and N = 8 individuals with APDS). Themes identified from HCPs interviews included symptoms, clinical manifestations, HRQoL impacts and treatments/management of APDS. The narrative account exercise identified similar themes, but with the addition to the journey to diagnosis. These themes were explored during the individual/caregiver interviews. Reported clinical manifestations and symptoms of APDS included susceptibility to infections, lymphoproliferation, gastrointestinal (GI) disorders, fatigue, bodily pain, and breathing difficulties. HRQoL impacts of living with APDS included negative impacts to daily activities, including work, education and social and leisure activities, physical functioning, as well as emotional well-being, such as concern for the future, and interpersonal relationships. Impacts to caregiver HRQoL included negative impacts to physical health, work, emotional well-being, interpersonal relationships and family life and holidays. The management of APDS included the use of healthcare services and medications including immunoglobulin replacement therapy (IRT), rapamycin, prophylactic antibiotics, leniolisib, as well as medical procedures due to complications. CONCLUSIONS: APDS has a high disease burden and there is an unmet need for licensed, more targeted treatments which modify disease progression. This study was the first to describe the day-to-day experience and HRQoL impact of APDS from the perspective of individuals living with the condition, caregivers and treating physicians.


Assuntos
Doenças da Imunodeficiência Primária , Qualidade de Vida , Humanos , Feminino , Masculino , Adulto , Cuidadores/psicologia , Pesquisa Qualitativa , Efeitos Psicossociais da Doença , Classe I de Fosfatidilinositol 3-Quinases/genética , Pessoa de Meia-Idade , Adolescente , Pessoal de Saúde/psicologia , Adulto Jovem , Doenças Genéticas Ligadas ao Cromossomo X/psicologia , Criança
3.
Cell ; 186(19): 4189-4203.e22, 2023 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-37633268

RESUMO

Thrombopoietin (THPO or TPO) is an essential cytokine for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Here, we report the 3.4 Å resolution cryoelectron microscopy structure of the extracellular TPO-TPO receptor (TpoR or MPL) signaling complex, revealing the basis for homodimeric MPL activation and providing a structural rationalization for genetic loss-of-function thrombocytopenia mutations. The structure guided the engineering of TPO variants (TPOmod) with a spectrum of signaling activities, from neutral antagonists to partial- and super-agonists. Partial agonist TPOmod decoupled JAK/STAT from ERK/AKT/CREB activation, driving a bias for megakaryopoiesis and platelet production without causing significant HSC expansion in mice and showing superior maintenance of human HSCs in vitro. These data demonstrate the functional uncoupling of the two primary roles of TPO, highlighting the potential utility of TPOmod in hematology research and clinical HSC transplantation.


Assuntos
Receptores de Trombopoetina , Trombopoetina , Animais , Humanos , Camundongos , Ciclo Celular , Microscopia Crioeletrônica , Receptores de Trombopoetina/genética , Trombopoese , Metilação de DNA
4.
Blood Adv ; 5(23): 4877-4889, 2021 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-34428275

RESUMO

Immune thrombocytopenia (ITP) is an acquired autoimmune condition characterized by both reduced platelet production and the destruction of functionally normal platelets by sustained attack from the immune system. However, the effect of prolonged ITP on the more immature hematopoietic progenitors remains an open area of investigation. By using a murine in vivo model of extended ITP, we revealed that ITP progression drives considerable progenitor expansion and bone marrow (BM) remodeling. Single-cell assays using Lin-Sca1+c-Kit+CD48-CD150+ long-term hematopoietic stem cells (LT-HSCs) revealed elevated LT-HSC activation and proliferation in vitro. However, the increased activation did not come at the expense of LT-HSC functionality as measured by in vivo serial transplantations. ITP progression was associated with considerable BM vasodilation and angiogenesis, as well as a twofold increase in the local production of CXCL12, a cytokine essential for LT-HSC function and BM homing expressed at high levels by LepR+ BM stromal cells. This was associated with a 1.5-fold increase in LepR+ BM stromal cells and a 5.5-fold improvement in progenitor homing to the BM. The increase in stromal cells was transient and reverted back to baseline after platelet count returned to normal, but the vasculature changes in the BM persisted. Together, our data demonstrate that LT-HSCs expand in response to ITP and that LT-HSC functionality during sustained hematopoietic stress is maintained through an adapting BM microenvironment.


Assuntos
Medula Óssea , Púrpura Trombocitopênica Idiopática , Animais , Hematopoese , Células-Tronco Hematopoéticas , Camundongos , Camundongos Endogâmicos C57BL
5.
Wellcome Open Res ; 6: 83, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34286101

RESUMO

Background: Polyparasitism is commonplace in countries where endemicity for multiple parasites exists, and studies in animal models of coinfection have made significant inroads into understanding the impact of often competing demands on the immune system. However, few studies have addressed how previous exposure to and treatment for one infection impacts a subsequent heterologous infection.   Methods: We used a C57BL/6 mouse model of drug-treated Leishmania donovani infection followed by experimental Plasmodium chabaudi AS malaria, focusing on hematological dysfunction as a common attribute of both infections. We measured parasite burden, blood parameters associated with anemia and thrombocytopenia, and serum thrombopoietin. In addition, we quantified macrophage iNOS expression through immunohistological analysis of the liver and spleen.   Results: We found that the thrombocytopenia and anemia that accompanies primary L. donovani infection was rapidly reversed following single dose AmBisome® treatment, along with multiple other markers associated with immune activation (including restoration of tissue microarchitecture and reduced macrophage iNOS expression). Compared to naive mice, mice cured of previous VL showed comparable albeit delayed clinical responses (including peak parasitemia and anemia) to P. chabaudi AS infection. Thrombocytopenia was also evident in these sequentially infected mice, consistent with a decrease in circulating levels of thrombopoietin. Architectural changes to the spleen were also comparable in sequentially infected mice compared to those with malaria alone. Conclusions: Our data suggest that in this sequential infection model, previously-treated VL has limited impact on the subsequent development of malaria, but this issue deserves further attention in models of more severe disease or through longitudinal population studies in humans.

6.
Platelets ; 32(6): 770-778, 2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34097561

RESUMO

Thrombopoietin (TPO) and its receptor, MPL, are the primary regulators of platelet production and critical for hematopoietic stem cell (HSC) maintenance. Since TPO was first cloned in 1994, the physiological and pathological roles of TPO and MPL have been well characterized, culminating in the first MPL agonists being approved for the treatment of chronic immune thrombocytopenia in 2008. Dysregulation of the TPO-MPL signaling axis contributes to the pathogenesis of hematological disorders: decreased expression or function results in severe thrombocytopenia progressing to bone marrow failure, while hyperactivation of MPL signaling, either by mutations in the receptor or associated Janus kinase 2 (JAK2), results in pathological myeloproliferation. Despite its importance, it was only recently that the long-running debate over the mechanism by which TPO binding activates MPL has been resolved. This review will cover key aspects of TPO and MPL structure and function and their importance in receptor activation, discuss how these are altered in hematological disorders and consider how a greater understanding could lead to the development of better-targeted and more efficacious therapies.


Assuntos
Plaquetas/metabolismo , Receptores de Trombopoetina/metabolismo , Humanos , Transdução de Sinais
7.
Blood Adv ; 5(6): 1627-1637, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33710338

RESUMO

Visceral leishmaniasis is an important yet neglected parasitic disease caused by infection with Leishmania donovani or L infantum. Disease manifestations include fever, weight loss, hepatosplenomegaly, immune dysregulation, and extensive hematological complications. Thrombocytopenia is a dominant hematological feature seen in both humans and experimental models, but the mechanisms behind this infection-driven thrombocytopenia remain poorly understood. Using a murine model of experimental visceral leishmaniasis (EVL), we demonstrated a progressive decrease in platelets from day 14 after infection, culminating in severe thrombocytopenia by day 28. Plasma thrombopoietin (TPO) levels were reduced in infected mice, at least in part because of the alterations in the liver microenvironment associated with granulomatous inflammation. Bone marrow (BM) megakaryocyte cytoplasmic maturation was significantly reduced. In addition to a production deficit, we identified significant increases in platelet clearance. L donovani-infected splenectomized mice were protected from thrombocytopenia compared with sham operated infected mice and had a greater response to exogenous TPO. Furthermore, infection led to higher levels of platelet opsonization and desialylation, both associated with platelet clearance in spleen and liver, respectively. Critically, these changes could be reversed rapidly by drug treatment to reduce parasite load or by administration of TPO agonists. In summary, our findings demonstrate that the mechanisms underpinning thrombocytopenia in EVL are multifactorial and reversible, with no obvious residual damage to the BM microenvironment.


Assuntos
Leishmaniose Visceral , Trombocitopenia , Animais , Modelos Animais de Doenças , Leishmaniose Visceral/complicações , Leishmaniose Visceral/tratamento farmacológico , Megacariócitos , Camundongos , Trombopoetina
8.
Proc Natl Acad Sci U S A ; 118(2)2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33384332

RESUMO

Thrombopoietin (TPO) and the TPO-receptor (TPO-R, or c-MPL) are essential for hematopoietic stem cell (HSC) maintenance and megakaryocyte differentiation. Agents that can modulate TPO-R signaling are highly desirable for both basic research and clinical utility. We developed a series of surrogate protein ligands for TPO-R, in the form of diabodies (DBs), that homodimerize TPO-R on the cell surface in geometries that are dictated by the DB receptor binding epitope, in effect "tuning" downstream signaling responses. These surrogate ligands exhibit diverse pharmacological properties, inducing graded signaling outputs, from full to partial TPO agonism, thus decoupling the dual functions of TPO/TPO-R. Using single-cell RNA sequencing and HSC self-renewal assays we find that partial agonistic diabodies preserved the stem-like properties of cultured HSCs, but also blocked oncogenic colony formation in essential thrombocythemia (ET) through inverse agonism. Our data suggest that dampening downstream TPO signaling is a powerful approach not only for HSC preservation in culture, but also for inhibiting oncogenic signaling through the TPO-R.


Assuntos
Receptores de Trombopoetina/metabolismo , Trombopoetina/metabolismo , Diferenciação Celular/fisiologia , Membrana Celular/metabolismo , Epitopos/imunologia , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Ligantes , Megacariócitos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Trombopoetina/imunologia , Receptores de Trombopoetina/fisiologia , Transdução de Sinais/fisiologia , Trombocitemia Essencial/metabolismo , Trombopoetina/fisiologia
9.
Front Physiol ; 11: 568087, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33041864

RESUMO

Platelets are specialized anucleate cells that play a major role in hemostasis following vessel injury. More recently, platelets have also been implicated in innate immunity and inflammation by directly interacting with immune cells and releasing proinflammatory signals. It is likely therefore that in certain pathologies, such as chronic parasitic infections and myeloid malignancies, platelets can act as mediators for hemostatic and proinflammatory responses. Fortunately, murine platelet function ex vivo is highly analogous to human, providing a robust model for functional comparison. However, traditional methods of studying platelet phenotype, function and activation status often rely on using large numbers of whole isolated platelet populations, which severely limits the number and type of assays that can be performed with mouse blood. Here, using cutting edge 3D quantitative phase imaging, holotomography, that uses optical diffraction tomography (ODT), we were able to identify and quantify differences in single unlabeled, live platelets with minimal experimental interference. We analyzed platelets directly isolated from whole blood of mice with either a JAK2V617F-positive myeloproliferative neoplasm (MPN) or Leishmania donovani infection. Image analysis of the platelets indicates previously uncharacterized differences in platelet morphology, including altered cell volume and sphericity, as well as changes in biophysical parameters such as refractive index (RI) and dry mass. Together, these data indicate that, by using holotomography, we were able to identify clear disparities in activation status and potential functional ability in disease states compared to control at the level of single platelets.

10.
Mol Cell ; 78(3): 477-492.e8, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32386542

RESUMO

Myelofibrosis is a severe myeloproliferative neoplasm characterized by increased numbers of abnormal bone marrow megakaryocytes that induce fibrosis, destroying the hematopoietic microenvironment. To determine the cellular and molecular basis for aberrant megakaryopoiesis in myelofibrosis, we performed single-cell transcriptome profiling of 135,929 CD34+ lineage- hematopoietic stem and progenitor cells (HSPCs), single-cell proteomics, genomics, and functional assays. We identified a bias toward megakaryocyte differentiation apparent from early multipotent stem cells in myelofibrosis and associated aberrant molecular signatures. A sub-fraction of myelofibrosis megakaryocyte progenitors (MkPs) are transcriptionally similar to healthy-donor MkPs, but the majority are disease specific, with distinct populations expressing fibrosis- and proliferation-associated genes. Mutant-clone HSPCs have increased expression of megakaryocyte-associated genes compared to wild-type HSPCs, and we provide early validation of G6B as a potential immunotherapy target. Our study paves the way for selective targeting of the myelofibrosis clone and illustrates the power of single-cell multi-omics to discover tumor-specific therapeutic targets and mediators of tissue fibrosis.


Assuntos
Hematopoese/fisiologia , Megacariócitos/patologia , Mielofibrose Primária/sangue , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular , Feminino , Regulação da Expressão Gênica , Hematopoese/genética , Células-Tronco Hematopoéticas/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Megacariócitos/fisiologia , Pessoa de Meia-Idade , Mutação , Receptores Imunológicos/genética , Análise de Célula Única/métodos
12.
Science ; 367(6478): 643-652, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32029621

RESUMO

Homodimeric class I cytokine receptors are assumed to exist as preformed dimers that are activated by ligand-induced conformational changes. We quantified the dimerization of three prototypic class I cytokine receptors in the plasma membrane of living cells by single-molecule fluorescence microscopy. Spatial and spatiotemporal correlation of individual receptor subunits showed ligand-induced dimerization and revealed that the associated Janus kinase 2 (JAK2) dimerizes through its pseudokinase domain. Oncogenic receptor and hyperactive JAK2 mutants promoted ligand-independent dimerization, highlighting the formation of receptor dimers as the switch responsible for signal activation. Atomistic modeling and molecular dynamics simulations based on a detailed energetic analysis of the interactions involved in dimerization yielded a mechanistic blueprint for homodimeric class I cytokine receptor activation and its dysregulation by individual mutations.


Assuntos
Carcinogênese/genética , Membrana Celular/química , Janus Quinase 2/química , Janus Quinase 2/genética , Multimerização Proteica , Receptores da Eritropoetina/química , Receptores da Somatotropina/química , Receptores de Trombopoetina/química , Substituição de Aminoácidos/genética , Células HeLa , Humanos , Janus Quinase 2/antagonistas & inibidores , Ligantes , Microscopia de Fluorescência , Modelos Moleculares , Mutação , Nitrilas , Fenilalanina/genética , Pirazóis/farmacologia , Pirimidinas , Transdução de Sinais , Imagem Individual de Molécula , Valina/genética
13.
J Tissue Viability ; 28(3): 133-138, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31085064

RESUMO

AIM: This study proposed to (1) develop a metric graduated colour tool and (2) demonstrate the effectiveness of the tool for use in the assessment of neonatal skin injuries. MATERIALS/METHODS: Findings from wound literature informed the metric graduated colour tool's development. Tool development included consideration of colours, size (comparative to neonatal skin injuries), cost, materials, feasibility and suitability for the neonatal clinical setting. Assessment of the tool's applicability with clinical images was then tested using digital cameras with specific evaluation of image sharpness and colour. Further evaluation was conducted within a case series of neonatal skin injuries. RESULTS: The metric graduated colour tool comprised of 15 colours, measures 60 mm, displays metric dimensions, and offers a discernible reference for clinical images and injury/wound bed comparison. Images collected appeared enhanced with clear wound edges compared to previous methods. Four neonates who acquired skin injuries were included in the case series for which the tool provided reliable metric and colour comparison of epidermal stripping, extravasation, birth injury, and pressure injury. When used to compare injury assessments for series subjects measurements of both increased and decreased severity were obtained. CONCLUSION: A metric and colour tool can be used in conjunction with digital photographs to enhance objective assessment of neonatal skin injuries/wounds. The metric and colour tool provides the foundation for vital skin injury assessment and documentation essentials including injury bed colour, size and consideration of depth of damage.


Assuntos
Pele/lesões , Lesões dos Tecidos Moles/classificação , Feminino , Humanos , Recém-Nascido , Masculino , Queensland , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Higiene da Pele , Lesões dos Tecidos Moles/diagnóstico , Pesos e Medidas/instrumentação , Pesos e Medidas/normas
14.
Proc Natl Acad Sci U S A ; 115(52): 13204-13209, 2018 12 26.
Artigo em Inglês | MEDLINE | ID: mdl-30530663

RESUMO

Cell communication is primarily regulated by secreted proteins, whose inhomogeneous secretion often indicates physiological disorder. Parallel monitoring of innate protein-secretion kinetics from individual cells is thus crucial to unravel systemic malfunctions. Here, we report a label-free, high-throughput method for parallel, in vitro, and real-time analysis of specific single-cell signaling using hyperspectral photonic crystal resonant technology. Heterogeneity in physiological thrombopoietin expression from individual HepG2 liver cells in response to platelet desialylation was quantified demonstrating how mapping real-time protein secretion can provide a simple, yet powerful approach for studying complex physiological systems regulating protein production at single-cell resolution.


Assuntos
Técnicas Biossensoriais/instrumentação , Processamento de Imagem Assistida por Computador/métodos , Rim/metabolismo , Fótons , Análise de Célula Única/métodos , Trombopoetina/metabolismo , Animais , Técnicas Biossensoriais/métodos , Células Cultivadas , Cricetinae , Células Hep G2 , Humanos , Transdução de Sinais
15.
Front Immunol ; 9: 2958, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30619317

RESUMO

Human visceral leishmaniasis, a parasitic disease of major public health importance in developing countries, is characterized by variable degrees of severity of anemia, but the mechanisms underlying this change in peripheral blood have not been thoroughly explored. Here, we used an experimental model of visceral leishmaniasis in C57BL/6 mice to explore the basis of anemia following infection with Leishmania donovani. 28 days post-infection, mice showed bone marrow dyserythropoiesis by myelogram, with a reduction of TER119+ CD71-/+ erythroblasts. Reduction of medullary erythropoiesis coincided with loss of CD169high bone marrow stromal macrophages and a reduction of CXCL12-expressing stromal cells. Although the spleen is a site of extramedullary erythropoiesis and erythrophagocytosis, splenectomy did not impact the extent of anemia or affect the repression of medullary hematopoiesis that was observed in infected mice. In contrast, these changes in bone marrow erythropoiesis were not evident in B6.Rag2-/- mice, but could be fully reconstituted by adoptive transfer of IFNγ-producing but not IFNγ-deficient CD4+ T cells, mimicking the expansion of IFNγ-producing CD4+ T cells that occurs during infection in wild type mice. Collectively, these data indicate that anemia during experimental murine visceral leishmaniasis can be driven by defects associated with the bone marrow erythropoietic niche, and that this represents a further example of CD4+ T cell-mediated immunopathology affecting hematopoietic competence.


Assuntos
Anemia/etiologia , Linfócitos T CD4-Positivos/imunologia , Eritropoese/imunologia , Leishmania donovani/imunologia , Leishmaniose Visceral/imunologia , Transferência Adotiva , Animais , Medula Óssea/fisiologia , Linfócitos T CD4-Positivos/transplante , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Humanos , Interferon gama/genética , Interferon gama/imunologia , Interferon gama/metabolismo , Leishmaniose Visceral/complicações , Leishmaniose Visceral/parasitologia , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Estromais/fisiologia
16.
Blood ; 124(26): 3956-63, 2014 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-25339357

RESUMO

The most frequent contributing factor in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) is the acquisition of a V617F mutation in Janus kinase 2 (JAK2) in hematopoietic stem cells (HSCs). Recent evidence has demonstrated that to drive MPN transformation, JAK2V617F needs to directly associate with a functional homodimeric type I cytokine receptor, suggesting that, although acquiring JAK2V617F may promote disease, there are additional cellular components necessary for MPN development. Here we show that loss of the thrombopoietin (TPO) receptor (MPL) significantly ameliorates MPN development in JAK2V617F(+) transgenic mice, whereas loss of TPO only mildly affects the disease phenotype. Specifically, compared with JAK2V617F(+) mice, JAK2V617F(+)Mpl(-/-) mice exhibited reduced thrombocythemia, neutrophilia, splenomegaly, and neoplastic stem cell pool. The importance of MPL is highlighted as JAK2V617FMpl(+/-) mice displayed a significantly reduced MPN phenotype, indicating that Mpl level may have a substantial effect on MPN development and severity. Splenomegaly and the increased neoplastic stem cell pool were retained in JAK2V617F(+)Tpo(-/-) mice, although thrombocytosis was reduced compared with JAK2V617F(+) mice. These results demonstrate that Mpl expression, but not Tpo, is fundamental in the development of JAK2V617F(+) MPNs, highlighting an entirely novel target for therapeutic intervention.


Assuntos
Células-Tronco Hematopoéticas/citologia , Janus Quinase 2/genética , Transtornos Mieloproliferativos/metabolismo , Receptores de Trombopoetina/genética , Animais , Células da Medula Óssea/citologia , Proliferação de Células , Citocinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Hematopoese , Heterozigoto , Homozigoto , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Trombopoetina/fisiologia , Células-Tronco/citologia
17.
Exp Hematol ; 42(6): 477-86.e4, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24607955

RESUMO

Thrombopoietin (TPO) is the primary regulator of platelet production, affecting cell survival, proliferation, and differentiation through binding to and stimulation of the cell surface receptor the cellular myeloproliferative leukemia virus oncogene (c-Mpl). Activating mutations in c-Mpl constitutively stimulate downstream signaling pathways, leading to aberrant hematopoiesis, and contribute to development of myeloproliferative neoplasms. Several studies have mapped the tyrosine residues within the cytoplasmic domain of c-Mpl that mediate these cellular signals; however, secondary signaling pathways are incompletely understood. In this study, we focused on c-Mpl tyrosine 591 (Y591). We found Y591 of wild-type c-Mpl to be phosphorylated in the presence of TPO. Additionally, eliminating Y591 phosphorylation by mutation to Phe resulted in decreased total receptor phosphorylation. Using a Src homology 2/phosphotyrosine-binding (SH2/PTB) domain binding microarray, we identified novel c-Mpl binding partners for phosphorylated Y591, including Src homology region 2 domain-containing phosphatase-1 (SHP-1), spleen tyrosine kinase (SYK) and Bruton's tyrosine kinase (BTK). The functional significance of binding partners was determined through small interfering RNA treatment of Ba/F3-Mpl cells, confirming that the increase in pERK1/2 resulting from removal of Y591 may be mediated by spleen tyrosine kinase. These findings identify a novel negative regulatory pathway that controls TPO-mediated signaling, advancing our understanding of the mechanisms required for successful maintenance of hematopoietic stem cells and megakaryocyte development.


Assuntos
Receptores de Trombopoetina/metabolismo , Transdução de Sinais/genética , Trombopoetina/fisiologia , Tirosina/metabolismo , Sequência de Aminoácidos , Western Blotting , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Dados de Sequência Molecular , Fosforilação , Receptores de Trombopoetina/genética
18.
Br J Haematol ; 165(2): 259-68, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24499199

RESUMO

In the two decades since its cloning, thrombopoietin (TPO) has emerged not only as a critical haematopoietic cytokine, but also serves as a great example of bench-to-bedside research. Thrombopoietin, produced by the liver, is the primary regulator of megakaryocyte progenitor expansion and differentiation. Additionally, as TPO is vital for the maintenance of haematopoietic stem cells, it can truly be described as a pan-haematopoietic cytokine. Since recombinant TPO became available, the molecular mechanisms of TPO function have been the subject of extensive research. Via its receptor, c-Mpl (also termed MPL), TPO activates a wide array of downstream signalling pathways, promoting cellular survival and proliferation. Due to its central, non-redundant role in haematopoiesis, alterations of both the hormone and its receptor contribute to human disease; congenital and acquired states of thrombocytosis and thrombocytopenia and aplastic anaemia as a result from dysregulated TPO expression or functional alterations of c-Mpl. With TPO mimetics now in clinical use, the story of this haematopoietic cytokine represents a great success for biomedical research.


Assuntos
Trombopoetina/fisiologia , Animais , Plaquetas/citologia , Plaquetas/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Receptores de Trombopoetina/genética , Receptores de Trombopoetina/metabolismo , Transdução de Sinais , Trombopoese/fisiologia , Trombopoetina/química , Trombopoetina/uso terapêutico
19.
Proc Natl Acad Sci U S A ; 111(6): 2295-300, 2014 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-24469804

RESUMO

The Janus kinase 2 (JAK2) V617F mutation is the primary pathogenic mutation in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs). Although thrombohemorrhagic incidents are the most common causes of morbidity and mortality in patients with MPNs, the events causing these clotting abnormalities remain unclear. To identify the cells responsible for the dysfunctional hemostasis, we used transgenic mice expressing JAK2V617F in specific lineages involved in thrombosis and hemostasis. When JAK2V617F was expressed in both hematopoietic and endothelial cells (ECs), the mice developed a significant MPN, characterized by thrombocytosis, neutrophilia, and splenomegaly. However, despite having significantly higher platelet counts than controls, these mice showed severely attenuated thrombosis following injury. Interestingly, platelet activation and aggregation in response to agonists was unaltered by JAK2V617F expression. Subsequent bone marrow transplants revealed the contribution of both endothelial and hematopoietic compartments to the attenuated thrombosis. Furthermore, we identified a potential mechanism for this phenotype through JAK2V617F-regulated inhibition of von Willebrand factor (VWF) function and/or secretion. JAK2V617F(+) mice display a condition similar to acquired von Willebrand syndrome, exhibiting significantly less high molecular weight VWF and reduced agglutination to ristocetin. These findings greatly advance our understanding of thrombohemorrhagic events in MPNs and highlight the critical role of ECs in the pathology of hematopoietic malignancies.


Assuntos
Transtornos da Coagulação Sanguínea/enzimologia , Endotélio Vascular/enzimologia , Janus Quinase 2/metabolismo , Transtornos Mieloproliferativos/complicações , Animais , Transtornos da Coagulação Sanguínea/complicações , Plaquetas/patologia , Camundongos , Camundongos Transgênicos , Receptor TIE-2/genética , Doenças de von Willebrand/genética
20.
Blood ; 123(7): 1059-68, 2014 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-24381227

RESUMO

Along with the most common mutation, JAK2V617F, several other acquired JAK2 mutations have now been shown to contribute to the pathogenesis of myeloproliferative neoplasms (MPNs). However, here we describe for the first time a germline mutation that leads to familial thrombocytosis that involves a residue other than Val617. The novel mutation JAK2R564Q, identified in a family with autosomal dominant essential thrombocythemia, increased cell growth resulting from suppression of apoptosis in Ba/F3-MPL cells. Although JAK2R564Q and JAK2V617F have similar levels of increased kinase activity, the growth-promoting effects of JAK2R564Q are much milder than those of JAK2V617F because of at least 2 counterregulatory mechanisms. Whereas JAK2V617F can escape regulation by the suppressor of cytokine signaling 3 and p27/Kip1, JAK2R564Q-expressing cells cannot. Moreover, JAK2R564Q-expressing cells are much more sensitive to the JAK inhibitor, ruxolitinib, than JAK2V617F-expressers, suggesting that lower doses of this drug may be effective in treating patients with MPNs associated with alternative JAK2 mutations, allowing many undesirable adverse effects to be avoided. This work provides a greater understanding of the cellular effects of a non-JAK2V617F, MPN-associated JAK2 mutation; provides insights into new treatment strategies for such patients; and describes the first case of familial thrombosis caused by a JAK2 residue other than Val617.


Assuntos
Mutação em Linhagem Germinativa , Janus Quinase 2/genética , Trombocitemia Essencial/genética , Adulto , Sequência de Aminoácidos , Substituição de Aminoácidos , Arginina/genética , Sequência de Bases , Criança , Feminino , Ácido Glutâmico/genética , Humanos , Janus Quinase 2/química , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Linhagem
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