RESUMO
Immune aplastic anemia (AA) is a severe blood disease characterized by T-lymphocyte- mediated stem cell destruction. Hematopoietic stem cell transplantation and immunosuppression are effective, but they entail costs and risks, and are not always successful. The Janus kinase (JAK) 1/2 inhibitor ruxolitinib (RUX) suppresses cytotoxic T-cell activation and inhibits cytokine production in models of graft-versus-host disease. We tested RUX in murine immune AA for potential therapeutic benefit. After infusion of lymph node (LN) cells mismatched at the major histocompatibility complex [C67BL/6 (B6)âCByB6F1], RUX, administered as a food additive (Rux-chow), attenuated bone marrow hypoplasia, ameliorated peripheral blood pancytopenia, preserved hematopoietic progenitors, and prevented mortality, when used either prophylactically or therapeutically. RUX suppressed the infiltration, proliferation, and activation of effector T cells in the bone marrow and mitigated Fas-mediated apoptotic destruction of target hematopoietic cells. Similar effects were obtained when Rux-chow was fed to C.B10 mice in a minor histocompatibility antigen mismatched (B6âC.B10) AA model. RUX only modestly suppressed lymphoid and erythroid hematopoiesis in normal and irradiated CByB6F1 mice. Our data support clinical trials of JAK/STAT inhibitors in human AA and other immune bone marrow failure syndromes.
Assuntos
Anemia Aplástica , Doenças da Medula Óssea , Pancitopenia , Camundongos , Humanos , Animais , Pancitopenia/patologia , Anemia Aplástica/patologia , Transtornos da Insuficiência da Medula Óssea/patologia , Medula Óssea/patologia , Doenças da Medula Óssea/patologia , Janus Quinase 1RESUMO
Immune aplastic anemia (AA) features somatic loss of HLA class I allele expression on bone marrow cells, consistent with a mechanism of escape from T-cell-mediated destruction of hematopoietic stem and progenitor cells. The clinical significance of HLA abnormalities has not been well characterized. We examined the somatic loss of HLA class I alleles and correlated HLA loss and mutation-associated HLA genotypes with clinical presentation and outcomes after immunosuppressive therapy in 544 AA patients. HLA class I allele loss was detected in 92 (22%) of the 412 patients tested, in whom there were 393 somatic HLA gene mutations and 40 instances of loss of heterozygosity. Most frequently affected was HLA-B*14:02, followed by HLA-A*02:01, HLA-B*40:02, HLA-B*08:01, and HLA-B*07:02. HLA-B*14:02, HLA-B*40:02, and HLA-B*07:02 were also overrepresented in AA. High-risk clonal evolution was correlated with HLA loss, HLA-B*14:02 genotype, and older age, which yielded a valid prediction model. In 2 patients, we traced monosomy 7 clonal evolution from preexisting clones harboring somatic mutations in HLA-A*02:01 and HLA-B*40:02. Loss of HLA-B*40:02 correlated with higher blood counts. HLA-B*07:02 and HLA-B*40:01 genotypes and their loss correlated with late-onset of AA. Our results suggest the presence of specific immune mechanisms of molecular pathogenesis with clinical implications. HLA genotyping and screening for HLA loss may be of value in the management of immune AA. This study was registered at clinicaltrials.gov as NCT00001964, NCT00061360, NCT00195624, NCT00260689, NCT00944749, NCT01193283, and NCT01623167.
Assuntos
Anemia Aplástica/genética , Genes MHC Classe I , Antígenos HLA-A/genética , Antígenos HLA-B/genética , Mutação , Adolescente , Adulto , Alelos , Anemia Aplástica/imunologia , Evolução Clonal , Feminino , Deleção de Genes , Expressão Gênica , Antígenos HLA-A/imunologia , Antígenos HLA-B/imunologia , Humanos , Imunidade , Perda de Heterozigosidade , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
We previously reported that granulocytic myeloid-derived suppressor cells (G-MDSCs) suppressed T-cell activation and attenuated bone marrow failure (BMF) in a minor histocompatibility (minor-H) antigen mismatched murine aplastic anemia (AA) model. In the current study, we tested the hypothesis that exosomes, a subset of extracellular vesicles, are responsible at least partially for G-MDSCs' therapeutic efficacy. Indeed, exosomes isolated from GMDSCs (G-MDSC-exos) suppressed CD4+ and CD8+ T-cell proliferation in vitro and mildly attenuated immune BMF in the minor-H mismatched AA model. G-MDSC-exos treatment significantly increased red blood cells, hemoglobin, and total bone marrow (BM) cells, and moderately reduced BM CD8+ T cells. G-MDSC-exos' effects were associated with upregulations in an array of lymphocyte-suppression-related miRNAs such as hsa-miR-142-5p, miR-19a-3p, and miR-19b-3p in both BM CD4+ and CD8+ T cells. We concluded that G-MDSC-exos attenuate immune BMF via modulating the delivery of immunosuppressive miRNAs into activated T lymphocytes.
Assuntos
Exossomos , MicroRNAs , Células Supressoras Mieloides , Pancitopenia , Camundongos , Animais , Linfócitos T CD8-Positivos , Modelos Animais de Doenças , Granulócitos , Imunossupressores/farmacologia , MicroRNAs/genética , Transtornos da Insuficiência da Medula ÓsseaRESUMO
Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase and central regulator of cell growth, differentiation, and survival. mTOR is commonly hyperactivated in a diverse number of cancers and critical roles for mTOR in regulating immune cell differentiation and function have been demonstrated. However, there is little work investigating the roles of mTOR in early B-cell development. Here we demonstrate that conditional disruption of mTOR in developing mouse B cells results in reduced pre-B-cell proliferation and survival, as well as a developmental block at the pre-B-cell stage, with a corresponding lack of peripheral B cells. Upon immunization with NP-CGG antigen, mice with Mtor conditional disruption in early B cells lost their ability to form germinal centers and produce specific antibodies. In competitive BM repopulation assays, donor BM cells from conditional knock-out mice were completely impaired in their ability to reconstitute B cells. Our data reveal the essential role of mTOR in early pre-B-cell development and survival.
Assuntos
Transdução de Sinais , Sirolimo , Animais , Linfócitos B/metabolismo , Diferenciação Celular , Ativação Linfocitária , Camundongos , Camundongos Knockout , Serina-Treonina Quinases TOR/metabolismoRESUMO
Pretreatment blood counts, particularly an absolute reticulocyte count ≥25×109/L, correlate with response to immunosuppressive therapy in severe aplastic anemia. In recent trials, eltrombopag combined with standard immunosuppressive therapy yielded superior responses than those to immunosuppressive therapy alone. Our single institution retrospective study aimed to elucidate whether historical predictors of response to immunosuppressive therapy alone were also associated with response to immunosuppressive therapy plus eltrombopag. We sought correlations of blood counts, thrombopoietin levels and the presence of paroxysmal nocturnal hemoglobinuria clones with both overall and complete responses in 416 patients with severe aplastic anemia, aged 2-82 years (median, 30 years), initially treated with immunosuppressive therapy plus eltrombopag between 2012 and 2019 (n=176) or with immunosuppressive therapy alone between 1999 and 2010 (n=240). Compared to non-responders, patients in the group of overall responders to immunosuppressive therapy plus eltrombopag had significantly higher pretreatment absolute reticulocyte counts, higher neutrophil counts and reduced thrombopoietin levels, as also observed for the group treated with immunosuppressive therapy alone. Addition of eltrombopag markedly improved the overall response in subjects with an absolute reticulocyte count between 10-30×109/L from 60% (54 of 90) to 91% (62 of 68). Absolute lymphocyte count correlated with complete response in the groups treated with immunosuppressive therapy with or without eltrombopag, especially in adolescents aged ≥10 years and adults, but the correlation was reversed in younger children. Platelet count and the presence of a paroxysmal nocturnal hemoglobinuria clone did not correlate with responses to immunosuppressive therapy. Blood counts remain the best predictors of response to nontransplant therapies in severe aplastic anemia. Addition of eltrombopag to immunosuppressive therapy shifted patients with a lower absolute reticulocyte count into a better prognostic category.
Assuntos
Anemia Aplástica , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Anemia Aplástica/diagnóstico , Anemia Aplástica/tratamento farmacológico , Benzoatos/uso terapêutico , Criança , Pré-Escolar , Humanos , Hidrazinas/uso terapêutico , Terapia de Imunossupressão , Imunossupressores/uso terapêutico , Pessoa de Meia-Idade , Pirazóis , Estudos Retrospectivos , Adulto JovemRESUMO
The role of mammalian target of rapamycin and its suppressor sirolimus in the regulation of hematopoietic stem and progenitor cells (HSPCs) is controversial. We show here that sirolimus enhanced regeneration of HSPCs in mice exposed to sublethal total body irradiation (TBI) and other regenerative stressors. Sorted Lin- CD150+ bone marrow cells from sirolimus-treated TBI mice had increased expression of c-Kit and other hematopoietic genes. HSPCs from sirolimus-treated TBI mice were functionally competent when tested by competitive engraftment in vivo. Postradiation regeneration of HSPCs in mice treated with sirolimus was accompanied by decreased γ-H2AX levels detected by flow cytometry and increased expression of DNA repair genes by quantitative polymerase chain reaction. Reduction of cell death and DNA damage post-radiation by sirolimus was associated with enhanced clearance of cellular reactive oxygen species (ROS) in HSPCs. Increased HSPC recovery with sirolimus was also observed in mice injected with hematoxic agents, busulfan and 5-fluorouracil. In contrast, sirolimus showed no effect on HSPCs in normal mice at steady state, but stimulated HSPC expansion in mice carrying the Wv mutation at the c-Kit locus. In human to mouse xenotransplantation, sirolimus enhanced engraftment of irradiated human CD34+ cells. In summary, our results are consistent with sirolimus' acceleration of HSPC recovery in response to hematopoietic stress, associated with reduced DNA damage and ROS. Sirolimus might have clinical application for the treatment and prevention of hematopoietic injury.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/fisiologia , Imunossupressores/farmacologia , Sirolimo/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Sobrevivência Celular/efeitos da radiação , Fluoruracila/toxicidade , Células-Tronco Hematopoéticas/efeitos da radiação , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia , Células-Tronco/efeitos da radiação , Irradiação Corporal Total/efeitos adversosRESUMO
The proinflammatory cytokine interferon-γ (IFN-γ) has been implicated in human hematopoietic stem and progenitor cell (HSPC) depletion in immune-mediated bone marrow failure syndromes. We show that IFN-γ specifically prevents full engagement of thrombopoietin (TPO), a primary positive regulator of HSPC survival, to its receptor (c-MPL) via steric occlusion of the low-affinity binding site, contributing to perturbation of TPO-induced signaling pathways and decreased survival of human HSPCs. Eltrombopag, a synthetic small molecule mimetic of TPO that interacts with c-MPL at a position distinct from the extracellular binding site of TPO, bypasses this inhibition, providing an explanation for its clinical activity in bone marrow failure, despite already elevated endogenous TPO levels. Thus, IFN-γ-mediated perturbation of TPO:c-MPL complex formation and the resulting inhibition of a critical pathway of growth factor cell signaling may represent a general mechanism by which IFN-γ impairs the function of human HSPCs. This understanding could have broad therapeutic implications for various disorders of chronic inflammation.
Assuntos
Benzoatos/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Hidrazinas/farmacologia , Interferon gama/metabolismo , Pirazóis/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células-Tronco Hematopoéticas/metabolismo , Xenoenxertos , Humanos , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptores de Trombopoetina/metabolismo , Transdução de Sinais/fisiologia , Trombopoetina/metabolismoRESUMO
BACKGROUND: Presently, there is no comprehensive analysis of the transcription regulation network in hematopoiesis. Comparison of networks arising from gene co-expression across species can facilitate an understanding of the conservation of functional gene modules in hematopoiesis. RESULTS: We used single-cell RNA sequencing to profile bone marrow from human and mouse, and inferred transcription regulatory networks in each species in order to characterize transcriptional programs governing hematopoietic stem cell differentiation. We designed an algorithm for network reconstruction to conduct comparative transcriptomic analysis of hematopoietic gene co-expression and transcription regulation in human and mouse bone marrow cells. Co-expression network connectivity of hematopoiesis-related genes was found to be well conserved between mouse and human. The co-expression network showed "small-world" and "scale-free" architecture. The gene regulatory network formed a hierarchical structure, and hematopoiesis transcription factors localized to the hierarchy's middle level. CONCLUSIONS: Transcriptional regulatory networks are well conserved between human and mouse. The hierarchical organization of transcription factors may provide insights into hematopoietic cell lineage commitment, and to signal processing, cell survival and disease initiation.
Assuntos
Hematopoese , Células-Tronco Hematopoéticas , Animais , Diferenciação Celular/genética , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Hematopoese/genética , Humanos , Camundongos , Análise de Sequência de RNARESUMO
Immune aplastic anaemia (AA) is caused by cytotoxic T lymphocytes (CTLs) that destroy haematopoietic stem and progenitor cells. Enhanced type 1 T helper (Th1) responses and reduced regulatory T cells (Tregs) are involved in the immune pathophysiology. CD24hi CD38hi regulatory B cells (Bregs) suppress CTLs and Th1 responses, and induce Tregs via interleukin 10 (IL-10). We investigated circulating B-cell subpopulations, including CD24hi CD38hi Bregs, as well as total B cells, CD4+ T cells, CD8+ T cells and natural killer cells in 104 untreated patients with severe and very severe AA, aged ≥18 years. All patients were treated with standard immunosuppressive therapy (IST) plus eltrombopag. CD24hi CD38hi Bregs were markedly reduced in patients with AA compared to healthy individuals, especially in very severe AA, but residual Bregs remained functional, capable of producing IL-10; total B-cell counts and the other B-cell subpopulations were similar to those of healthy individuals. CD24hi CD38hi Bregs did not correlate with responses to IST, and they recovered to levels present in healthy individuals after therapy. Mature naïve B-cell counts were unexpectedly associated with IST response. Markedly reduced CD24hi CD38hi Bregs, especially in very severe AA, with recovery after IST suggest Breg deficits may contribute to the pathophysiology of immune AA.
Assuntos
ADP-Ribosil Ciclase 1/análise , Anemia Aplástica/sangue , Antígenos CD19/análise , Subpopulações de Linfócitos B/patologia , Linfócitos B Reguladores/patologia , Antígeno CD24/análise , Linfopenia/etiologia , Glicoproteínas de Membrana/análise , Adolescente , Adulto , Idoso , Anemia Aplástica/complicações , Anemia Aplástica/tratamento farmacológico , Anemia Aplástica/patologia , Soro Antilinfocitário/uso terapêutico , Subpopulações de Linfócitos B/química , Linfócitos B Reguladores/química , Benzoatos/uso terapêutico , Medula Óssea/patologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Ciclosporina/uso terapêutico , Feminino , Humanos , Hidrazinas/uso terapêutico , Imunossupressores/uso terapêutico , Interleucina-10/biossíntese , Interleucina-10/genética , Células Matadoras Naturais/patologia , Contagem de Linfócitos , Linfopenia/sangue , Linfopenia/patologia , Masculino , Pessoa de Meia-Idade , Pirazóis/uso terapêutico , Receptores de Trombopoetina/agonistas , Adulto JovemRESUMO
BACKGROUND: Acquired aplastic anemia results from immune-mediated destruction of bone marrow. Immunosuppressive therapies are effective, but reduced numbers of residual stem cells may limit their efficacy. In patients with aplastic anemia that was refractory to immunosuppression, eltrombopag, a synthetic thrombopoietin-receptor agonist, led to clinically significant increases in blood counts in almost half the patients. We combined standard immunosuppressive therapy with eltrombopag in previously untreated patients with severe aplastic anemia. METHODS: We enrolled 92 consecutive patients in a prospective phase 1-2 study of immunosuppressive therapy plus eltrombopag. The three consecutively enrolled cohorts differed with regard to the timing of initiation and the duration of the eltrombopag regimen (cohort 1 received eltrombopag from day 14 to 6 months, cohort 2 from day 14 to 3 months, and cohort 3 from day 1 to 6 months). The cohorts were analyzed separately. The primary outcome was complete hematologic response at 6 months. Secondary end points included overall response, survival, relapse, and clonal evolution to myeloid cancer. RESULTS: The rate of complete response at 6 months was 33% in cohort 1, 26% in cohort 2, and 58% in cohort 3. The overall response rates at 6 months were 80%, 87%, and 94%, respectively. The complete and overall response rates in the combined cohorts were higher than in our historical cohort, in which the rate of complete response was 10% and the overall response rate was 66%. At a median follow-up of 2 years, the survival rate was 97%; one patient died during the study from a nonhematologic cause. Marked increases in bone marrow cellularity, CD34+ cell number, and frequency of early hematopoietic progenitors were noted. Rates of relapse and clonal evolution were similar to our historical experience. Severe rashes occurred in two patients, resulting in the early discontinuation of eltrombopag. CONCLUSIONS: The addition of eltrombopag to immunosuppressive therapy was associated with markedly higher rates of hematologic response among patients with severe aplastic anemia than in a historical cohort. (Funded by the National Heart, Lung, and Blood Institute; ClinicalTrials.gov number, NCT01623167 .).
Assuntos
Anemia Aplástica/tratamento farmacológico , Benzoatos/uso terapêutico , Fármacos Hematológicos/uso terapêutico , Hidrazinas/uso terapêutico , Imunossupressores/uso terapêutico , Pirazóis/uso terapêutico , Receptores de Trombopoetina/agonistas , Adolescente , Adulto , Idoso , Antígenos CD34 , Soro Antilinfocitário/uso terapêutico , Benzoatos/efeitos adversos , Contagem de Células , Ciclosporina/uso terapêutico , Quimioterapia Combinada , Feminino , Fármacos Hematológicos/efeitos adversos , Humanos , Hidrazinas/efeitos adversos , Terapia de Imunossupressão , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Pirazóis/efeitos adversos , Adulto JovemRESUMO
In humans, bone marrow (BM) failure syndromes, both constitutional and acquired, predispose to myeloid malignancies. We have modeled acquired immune aplastic anemia, the paradigmatic disease of these syndromes, in the mouse by infusing lymph node cells from specific pathogen-free (SPF) CD45.1 congenic C57BL/6 (B6) donors into hybrid CByB6F1 recipients housed either in conventional (CVB) or SPF facilities. The severity of BM damage was reduced in CVB recipients; they also had reduced levels of CD44+ CD62L- effector memory T cells, reduced numbers of donor-type CD44+ T cells, and reduced expansion of donor-type CD8 T cells carrying T-cell receptor ß-variable regions 07, 11, and 17. Analyses of fecal samples through 16S ribosomal RNA amplicon sequencing revealed greater gut microbial alpha diversity in CVB mice relative to that of SPF mice. Thus, the presence of a broader spectrum of gut microorganisms in CVB-housed CByB6F1 could have primed recipient animal's immune system leading to suppression of allogeneic donor T-cell activation and expansion and attenuation of host BM destruction. These results suggest the potential benefit of diverse gut microbiota in patients receiving BM transplants.
Assuntos
Anemia Aplástica/terapia , Transplante de Medula Óssea/métodos , Medula Óssea/imunologia , Microbioma Gastrointestinal/imunologia , Linfócitos T/imunologia , Anemia Aplástica/imunologia , Anemia Aplástica/patologia , Animais , Medula Óssea/patologia , Fezes/microbiologia , Receptores de Hialuronatos/imunologia , Receptores de Hialuronatos/metabolismo , Memória Imunológica/imunologia , Ativação Linfocitária/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Organismos Livres de Patógenos Específicos , Linfócitos T/metabolismo , Linfócitos T/transplante , Imunologia de Transplantes , Transplante HomólogoRESUMO
Interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) have been implicated historically in the immune pathophysiology of aplastic anemia (AA) and other bone marrow (BM) failure syndromes. We recently defined the essential roles of IFN-γ produced by donor T cells and the IFN-γ receptor in the host in murine immune-mediated BM failure models. TNF-α has been assumed to function similarly to IFN-γ. We used our murine models and mice genetically deficient in TNF-α or TNF-α receptors (TNF-αRs) to establish an analogous mechanism. Unexpectedly, infusion of TNF-α-/- donor lymph node (LN) cells into CByB6F1 recipients or injection of FVB LN cells into TNF-αR-/- recipients both induced BM failure, with concurrent marked increases in plasma IFN-γ and TNF-α levels. Surprisingly, in TNF-α-/- recipients, BM damage was attenuated, suggesting that TNF-α of host origin was essential for immune destruction of hematopoiesis. Depletion of host macrophages before LN injection reduced T-cell IFN-γ levels and reduced BM damage, whereas injection of recombinant TNF-α into FVB-LN cell-infused TNF-α-/- recipients increased T-cell IFN-γ expression and accelerated BM damage. Furthermore, infusion of TNF-αR-/- donor LN cells into CByB6F1 recipients reduced BM T-cell infiltration, suppressed T-cell IFN-γ production, and alleviated BM destruction. Thus, TNF-α from host macrophages and TNF-αR expressed on donor effector T cells were critical in the pathogenesis of murine immune-mediated BM failure, acting by modulation of IFN-γ secretion. In AA patients, TNF-α-producing macrophages in the BM were more frequent than in healthy controls, suggesting the involvement of this cytokine and these cells in human disease.
Assuntos
Anemia Aplástica/imunologia , Doenças da Medula Óssea/imunologia , Hemoglobinúria Paroxística/imunologia , Macrófagos/imunologia , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/imunologia , Aloenxertos , Anemia Aplástica/genética , Anemia Aplástica/patologia , Anemia Aplástica/terapia , Animais , Doenças da Medula Óssea/genética , Doenças da Medula Óssea/patologia , Doenças da Medula Óssea/terapia , Transtornos da Insuficiência da Medula Óssea , Transplante de Medula Óssea , Hemoglobinúria Paroxística/genética , Hemoglobinúria Paroxística/patologia , Hemoglobinúria Paroxística/terapia , Humanos , Interferon gama/genética , Interferon gama/imunologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Linfócitos T/patologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
Myelodysplastic syndromes (MDS) are a group of clonal myeloid disorders characterized by cytopenia and a propensity to develop acute myeloid leukemia (AML). The management of lower-risk (LR) MDS with persistent cytopenias remains suboptimal. Eltrombopag (EPAG), a thrombopoietin receptor agonist, can improve platelet counts in LR-MDS and tri-lineage hematopoiesis in aplastic anemia (AA). We conducted a phase 2 dose modification study to investigate the safety and efficacy of EPAG in LR-MDS. EPAG dose was escalated from 50 mg/day, to a maximum of 150 mg/day over a period of 16 weeks. The primary efficacy endpoint was hematologic response at 16-20 weeks. Eleven of 25 (44%) patients responded; five and six patients had uni- or bi-lineage hematologic responses, respectively. The predictors of response were presence of a PNH clone, marrow hypocellularity, thrombocytopenia with or without other cytopenia, and elevated plasma thrombopoietin levels at study entry. The safety profile was consistent with previous EPAG studies in AA; no patients discontinued drug due to adverse events. Three patients developed reversible grade-3 liver toxicity and one patient had increased reticulin fibrosis. Ten patients discontinued EPAG after achieving a robust response (median time 16 months); four of them reinitiated EPAG due to declining counts, and all attained a second robust response. Six patients had disease progression not associated with expansion of mutated clones and no patient progressed to AML on study. In conclusion, EPAG was well-tolerated and effective in restoring hematopoiesis in patients with low to intermediate-1 risk MDS. This study was registered at clinicaltrials.gov as #NCT00932156.
Assuntos
Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Benzoatos/efeitos adversos , Hematopoese , Humanos , Hidrazinas/efeitos adversos , Leucemia Mieloide Aguda/tratamento farmacológico , Síndromes Mielodisplásicas/tratamento farmacológico , PirazóisRESUMO
Specific-pathogen-free (SPF) mice have improved hematopoietic characteristics relative to germ-free mice, however, it is not clear whether improvements in hematopoietic traits will continue when the level of microorganism exposure is further increased. We co-housed SPF C57BL/6 mice in a conventional facility (CVT) and found a significant increase in gut microbiota diversity along with increased levels of myeloid cells and T cells, especially effector memory T cells. Through single cell RNA sequencing of sorted KL (c-Kit+Lin-) cells, we imputed a decline in long-term hematopoietic stem cells and an increase in granulocyte-monocyte progenitors in CVT mice with up-regulation of genes associated with cell survival. Bone marrow transplantation through competitive repopulation revealed a significant increase in KSL (c-Kit+Sca-1+Lin-) cell reconstitution in recipients of CVT donor cells which occurred when donors were co-housed for both one and twelve months. However, there was minimal to no gain in mature blood cell engraftment in recipients of CVT donor cells relative to those receiving SPF donor cells. We conclude that co-housing SPF mice with mice born in a conventional facility increased gut microbiota diversity, augmented myeloid cell production and T cell activation, stimulated KSL cell reconstitution, and altered hematopoietic gene expression.
Assuntos
Bactérias/classificação , Perfilação da Expressão Gênica/métodos , Hematopoese , Células Mieloides/metabolismo , Análise de Sequência de RNA/métodos , Linfócitos T/metabolismo , Animais , Bactérias/genética , Bactérias/isolamento & purificação , Transplante de Medula Óssea , Microbioma Gastrointestinal , Regulação da Expressão Gênica , Abrigo para Animais , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Filogenia , Análise de Célula Única , Organismos Livres de Patógenos EspecíficosRESUMO
Cancer cells frequently exhibit chromosomal abnormalities. Specific cytogenetic aberrations often are predictors of outcome, especially in hematologic neoplasms, such as monosomy 7 in myeloid malignancies. The functional consequences of aneuploidy at the cellular level are difficult to assess because of a lack of convenient markers to distinguish abnormal from diploid cells. We performed single-cell RNA sequencing (scRNA-seq) to study hematopoietic stem and progenitor cells from the bone marrow of 4 healthy donors and 5 patients with bone marrow failure and chromosome gain or loss. In total, transcriptome sequences were obtained from 391 control cells and 588 cells from patients. We characterized normal hematopoiesis as binary differentiation from stem cells to erythroid and myeloid-lymphoid pathways. Aneuploid cells were distinguished from diploid cells in patient samples by computational analyses of read fractions and gene expression of individual chromosomes. We confirmed assignment of aneuploidy to individual cells quantitatively, by copy-number variation, and qualitatively, by loss of heterozygosity. When we projected patients' single cells onto the map of normal hematopoiesis, diverse patterns were observed, broadly reflecting clinical phenotypes. Patients' monosomy 7 cells showed downregulation of genes involved in immune response and DNA damage checkpoint and apoptosis pathways, which may contribute to the clonal expansion of monosomy 7 cells with accumulated gene mutations. scRNA-seq is a powerful technique through which to infer the functional consequences of chromosome gain and loss and explore gene targets for directed therapy.
Assuntos
Aneuploidia , Células-Tronco Hematopoéticas , Análise de Sequência de RNA , Análise de Célula Única/métodos , Transcriptoma/genética , Adulto , Células da Medula Óssea , Doenças da Medula Óssea/genética , Doenças da Medula Óssea/patologia , Estudos de Casos e Controles , Deleção Cromossômica , Transtornos Cromossômicos/genética , Cromossomos Humanos Par 7 , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Long noncoding RNAs (lncRNAs) are regulators of cell differentiation and development. The lncRNA transcriptome in human hematopoietic stem and progenitor cells is not comprehensively defined. We investigated lncRNAs in 979 human bone marrow-derived CD34+ cells by single cell RNA sequencing followed by de novo transcriptome reconstruction. We identified 3,173 lncRNAs in total, among which 2,365 were previously unknown, and we characterized lncRNA stem, differentiation, and maturation signatures. lncRNA expression exhibited high cell-to-cell variation, which was only apparent in single cell analysis. lncRNA expression followed a lineage-specific and highly dynamic pattern during early hematopoiesis. lncRNAs in hematopoietic cells closely correlated with protein-coding genes of known functions in the regulation of hematopoiesis and cell fate decisions, and the potential regulatory roles of lncRNAs in hematopoiesis were imputed by projection from protein-coding genes with a "guilt-by-association" approach. We characterized lncRNAs preferentially expressed in hematopoietic stem cells and in various downstream differentiated lineage progenitors. We also profiled lncRNA expression in single cells from patients with myelodysplastic syndromes and in aneuploid cells in particular. Our study provides a global view of lncRNAs in human hematopoietic stem and progenitor cells. We observed a highly ordered pattern of lncRNA expression and participation in regulation of early hematopoiesis, and coordinate aberrant messenger RNA and lncRNA transcriptomes in dysplastic hematopoiesis. (Registered at clinicaltrials.gov with identifiers: 00001620, 00001397).
Assuntos
Biomarcadores Tumorais/genética , Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Síndromes Mielodisplásicas/genética , RNA Longo não Codificante/genética , Análise de Célula Única/métodos , Transcriptoma , Medula Óssea/patologia , Diferenciação Celular , Células Cultivadas , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Hematopoese , Células-Tronco Hematopoéticas/citologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Síndromes Mielodisplásicas/patologiaAssuntos
Ciclosporina , Nitrilas , Pirazóis , Pirimidinas , Pirazóis/uso terapêutico , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Pirimidinas/uso terapêutico , Animais , Camundongos , Ciclosporina/uso terapêutico , Ciclosporina/administração & dosagem , Quimioterapia Combinada , Transtornos da Insuficiência da Medula Óssea/tratamento farmacológico , Modelos Animais de DoençasRESUMO
Aplastic anemia (AA) is the most serious non-malignant blood disorder in Pakistan, ranked second in prevalence, after thalassemia. We investigated various epidemiological, clinical, and genetic factors of AA in a Pakistani cohort of 214 patients reporting at our hospital between June 2014 and December 2015. A control group of 214 healthy subjects was included for comparison of epidemiological and clinical features. Epidemiological data revealed 2.75-fold higher frequency of AA among males. A single peak of disease onset was observed between ages 10 and 29 years followed by a steady decline. AA was strongly associated with lower socioeconomic profile, rural residence, and high rate of consanguineous marriages. Serum granulocyte colony-stimulating factor and thrombopoietin levels were significantly elevated in AA patients, compared to healthy controls (P < 0.0001), while there was no statistical significance in other nine cytokine levels screened. Allele frequencies of DRB1*15 (56.8%) and DQB1*06 (70.3%) were predominantly high in AA patients. Ten mutations were found in TERT and TERC genes, including two novel mutations (Val526Ala and Val777Met) in exons 3 and 7 of TERT gene. Despite specific features of the AA cohort, this study suggests that epidemiologic and etiologic factors as well as host genetic predisposition exclusively or cooperatively trigger AA in Pakistan.
Assuntos
Anemia Aplástica , Mutação de Sentido Incorreto , Adolescente , Adulto , Idade de Início , Substituição de Aminoácidos , Anemia Aplástica/sangue , Anemia Aplástica/epidemiologia , Anemia Aplástica/genética , Criança , Feminino , Frequência do Gene , Fator Estimulador de Colônias de Granulócitos/sangue , Fator Estimulador de Colônias de Granulócitos/genética , Cadeias beta de HLA-DQ/sangue , Cadeias beta de HLA-DQ/genética , Cadeias HLA-DRB1/sangue , Cadeias HLA-DRB1/genética , Humanos , Masculino , Pessoa de Meia-Idade , Paquistão/epidemiologia , Fatores Sexuais , Fatores Socioeconômicos , Telomerase/sangue , Telomerase/genética , Trombopoetina/sangue , Trombopoetina/genéticaRESUMO
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired disorder originating from hematopoietic stem cells and is a life-threating disease characterized by intravascular hemolysis, bone marrow (BM) failure, and venous thrombosis. The etiology of PNH is a somatic mutation in the phosphatidylinositol glycan class A gene (PIG-A) on the X chromosome, which blocks synthesis of the glycolipid moiety and causes deficiency in GPI-anchored proteins. PNH is closely related to aplastic anemia, in which T cells mediate destruction of BM. To identify aberrant molecular mechanisms involved in immune targeting of hematopoietic stem cells in BM, we applied RNA-seq to examine the transcriptome of T cell subsets (CD4+ naive, CD4+ memory, CD8+ naive, and CD8+ memory) from PNH patients and healthy control subjects. Differentially expressed gene analysis in four different T cell subsets from PNH and healthy control subjects showed distinct transcriptional profiles, depending on the T cell subsets. By pathway analysis, we identified novel signaling pathways in T cell subsets from PNH, including increased gene expression involved in TNFR, IGF1, NOTCH, AP-1, and ATF2 pathways. Dysregulation of several candidate genes (JUN, TNFAIP3, TOB1, GIMAP4, GIMAP6, TRMT112, NR4A2, CD69, and TNFSF8) was validated by quantitative real-time RT-PCR and flow cytometry. We have demonstrated molecular signatures associated with positive and negative regulators in T cells, suggesting novel pathophysiologic mechanisms in PNH. These pathways may be targets for new strategies to modulate T cell immune responses in BM failure.