RESUMO
Transcription factor RUNX1 is a master regulator of hematopoiesis and megakaryopoiesis. RUNX1 haplodeficiency (RHD) is associated with thrombocytopenia and platelet granule deficiencies and dysfunction. Platelet profiling of our study patient with RHD showed decreased expression of RAB31, a small GTPase whose cell biology in megakaryocytes (MKs)/platelets is unknown. Platelet RAB31 messenger RNA was decreased in the index patient and in 2 additional patients with RHD. Promoter-reporter studies using phorbol 12-myristate 13-acetate-treated megakaryocytic human erythroleukemia cells revealed that RUNX1 regulates RAB31 via binding to its promoter. We investigated RUNX1 and RAB31 roles in endosomal dynamics using immunofluorescence staining for markers of early endosomes (EEs; early endosomal autoantigen 1) and late endosomes (CD63)/multivesicular bodies. Downregulation of RUNX1 or RAB31 (by small interfering RNA or CRISPR/Cas9) showed a striking enlargement of EEs, partially reversed by RAB31 reconstitution. This EE defect was observed in MKs differentiated from a patient-derived induced pluripotent stem cell line (RHD-iMKs). Studies using immunofluorescence staining showed that trafficking of 3 proteins with distinct roles (von Willebrand factor [VWF], a protein trafficked to α-granules; epidermal growth factor receptor; and mannose-6-phosphate) was impaired at the level of EE on downregulation of RAB31 or RUNX1. There was loss of plasma membrane VWF in RUNX1- and RAB31-deficient megakaryocytic human erythroleukemia cells and RHD-iMKs. These studies provide evidence that RAB31 is downregulated in RHD and regulates megakaryocytic vesicle trafficking of 3 major proteins with diverse biological roles. EE defect and impaired vesicle trafficking is a potential mechanism for the α-granule defects observed in RUNX1 deficiency.
Assuntos
Leucemia Eritroblástica Aguda , Megacariócitos , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Receptores ErbB/metabolismo , Humanos , Megacariócitos/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , Fator de von Willebrand/metabolismoRESUMO
BACKGROUND: Identification of biomarkers associated with immune-mediated diseases in 22q11.2 deletion syndrome is an evolving field. OBJECTIVES: We sought to use a carefully phenotyped cohort to study immune parameters associated with autoimmunity and atopy in 22q11.2 deletion syndrome to define biomarkers associated with immune-mediated disease in this syndrome. METHODS: Chart review validated autoimmune disease and atopic condition diagnoses. Laboratory data were extracted for each subcohort and plotted according to age. A random-effects model was used to define statistical significance. RESULTS: CD19, CD4, and CD4/45RA lymphocyte populations were not different from the general cohort for patients with atopic conditions. CD4/45RA T cells were significantly lower in the subjects with immune thrombocytopenia compared with the general cohort, and CD4 T-cell counts were lower in patients with autoimmune thyroid disease. CONCLUSIONS: The mechanisms of autoimmunity in cytopenias may be distinct from those of solid-organ autoimmunity in 22q11.2 deletion syndrome. This study identifies potential biomarkers for risk stratification among commonly obtained laboratory studies.
Assuntos
Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/imunologia , Síndrome de DiGeorge/imunologia , Hipersensibilidade Imediata/imunologia , Adolescente , Adulto , Contagem de Linfócito CD4 , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Fenótipo , Adulto JovemRESUMO
The last 10 years have seen an explosion in the amount of data available through next-generation sequencing. These data are advancing quickly, and this pace makes it difficult for most practitioners to easily keep up with all of the new information. Complicating this understanding is sometimes conflicting information about variant pathogenicity or even about the role of some genes in the pathogenesis of disease. The more widespread clinical use of sequencing has expanded phenotypes, including the identification of mild phenotypes associated with previously serious disease, such as with some variants in RUNX1, MYH9, ITG2A, and others. Several organizations have taken up the task of cataloging and systematically evaluating genes and variants using a standardized approach and making the data publicly available so that others can benefit from their gene/variant curation. The efforts in testing for hereditary hemorrhagic, thrombotic, and platelet disorders have been led by the International Society on Thrombosis and Haemostasis Scientific Standardization Committee on Genomics in Thrombosis and Hemostasis, the American Society of Hematology, and the National Institutes of Health National Human Genome Research Institute Clinical Genome Resource. This article outlines current efforts to improve the interpretation of genetic testing and the role of standardizing and disseminating information. By assessing the strength of gene-disease associations, standardizing variant curation guidelines, sharing genomic data among expert members, and incorporating data from existing disease databases, the number of variants of uncertain significance will decrease, thereby improving the value of genetic testing as a diagnostic tool.
Assuntos
Transtornos Plaquetários , Doenças Genéticas Inatas , Testes Genéticos , Hemorragia , Sequenciamento de Nucleotídeos em Larga Escala , Trombose , Adolescente , Transtornos Plaquetários/diagnóstico , Transtornos Plaquetários/genética , Feminino , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Hemorragia/diagnóstico , Hemorragia/genética , Humanos , Trombose/diagnóstico , Trombose/genéticaRESUMO
BACKGROUND: Although chiefly a B-lymphocyte disorder, several research groups have identified common variable immunodeficiency (CVID) subjects with numeric and/or functional TH cell alterations. The causes, interrelationships, and consequences of CVID-associated CD4+ T-cell derangements to hypogammaglobulinemia, autoantibody production, or both remain unclear. OBJECTIVE: We sought to determine how circulating CD4+ T cells are altered in CVID subjects with autoimmune cytopenias (AICs; CVID+AIC) and the causes of these derangements. METHODS: Using hypothesis-generating, high-dimensional single-cell analyses, we created comprehensive phenotypic maps of circulating CD4+ T cells. Differences between subject groups were confirmed in a large and genetically diverse cohort of CVID subjects (n = 69) by using flow cytometry, transcriptional profiling, multiplex cytokine/chemokine detection, and a suite of in vitro functional assays measuring naive T-cell differentiation, B-cell/T-cell cocultures, and regulatory T-cell suppression. RESULTS: Although CD4+ TH cell profiles from healthy donors and CVID subjects without AICs were virtually indistinguishable, T cells from CVID+AIC subjects exhibited follicular features as early as thymic egress. Follicular skewing correlated with IgA deficiency-associated endotoxemia and endotoxin-induced expression of activin A and inducible T-cell costimulator ligand. The resulting enlarged circulating follicular helper T-cell population from CVID+AIC subjects provided efficient help to receptive healthy donor B cells but not unresponsive CVID B cells. Despite this, circulating follicular helper T cells from CVID+AIC subjects exhibited aberrant transcriptional profiles and altered chemokine/cytokine receptor expression patterns that interfered with regulatory T-cell suppression assays and were associated with autoantibody production. CONCLUSIONS: Endotoxemia is associated with early commitment to the follicular T-cell lineage in IgA-deficient CVID subjects, particularly those with AICs.
Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Imunodeficiência de Variável Comum/imunologia , Endotoxemia/imunologia , Deficiência de IgA/imunologia , Linfócitos T Reguladores/imunologia , Adolescente , Adulto , Linfócitos B/patologia , Criança , Pré-Escolar , Imunodeficiência de Variável Comum/patologia , Endotoxemia/patologia , Feminino , Humanos , Deficiência de IgA/patologia , Masculino , Pessoa de Meia-Idade , Linfócitos T Reguladores/patologiaRESUMO
Thrombocytopenia is a significant complication of chemotherapy and radiation therapy. Platelet factor 4 (PF4; CXCL4) is a negative paracrine of megakaryopoiesis. We have shown that PF4 levels are inversely related to steady-state platelet counts, and to the duration and severity of chemotherapy- and radiation-induced thrombocytopenia (CIT and RIT, respectively). Murine studies suggest that blocking the effect of PF4 improves megakaryopoiesis, raising nadir platelet counts and shortening the time to platelet count recovery. We examined the ability of 2-O, 3-O desulfated heparin (ODSH), a heparin variant with little anticoagulant effects, to neutralize PF4's effects on megakaryopoiesis. Using megakaryocyte colony assays and liquid cultures, we show that ODSH restored megakaryocyte proliferation in PF4-treated Cxcl4-/- murine and human CD34+-derived megakaryocyte cultures (17.4% megakaryocyte colonies, P < .01 compared with PF4). In murine CIT and RIT models, ODSH, started 24 hours after injury, was examined for the effect on hematopoietic recovery demonstrating higher platelet count nadirs (9% ± 5% treated vs 4% ± 4% control) and significantly improved survival in treated animals (73% treated vs 36% control survival). Treatment with ODSH was able to reduce intramedullary free PF4 concentrations by immunohistochemical analysis. In summary, ODSH mitigated CIT and RIT in mice by neutralizing the intramedullary negative paracrine PF4. ODSH, already in clinical trials in humans as an adjuvant to chemotherapy, may be an important, clinically relevant therapeutic for CIT and RIT.
Assuntos
Heparina/análogos & derivados , Trombocitopenia/tratamento farmacológico , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Heparina/farmacologia , Heparina/uso terapêutico , Humanos , Megacariócitos/citologia , Camundongos , Contagem de Plaquetas , Fator Plaquetário 4/sangue , Fator Plaquetário 4/efeitos dos fármacos , Fator Plaquetário 4/farmacologia , Trombocitopenia/induzido quimicamente , Trombocitopenia/etiologia , TrombopoeseRESUMO
This review will discuss how 2 common and morbid conditions, renal disease and liver disease, alter platelet number and function. It will review the impact of thrombocytopenia on bleeding complications in patients with these disorders and whether the low platelet count actually correlates with bleeding risk. Emerging data also suggest that platelets are much more than bystanders in both renal and liver disease, but instead play an active role in the pathobiology of these disorders. This review will briefly cover the emerging information on novel roles of platelets in the biology of renal and liver disease.