Publications by authors named "Xingmin Feng"

58 Publications

Comprehensive network modeling from single cell RNA sequencing of human and mouse reveals well conserved transcription regulation of hematopoiesis.

BMC Genomics 2020 Dec 29;21(Suppl 11):849. Epub 2020 Dec 29.

Hematopoiesis and Bone Marrow Failure Laboratory, Hematology Branch, NHLBI, National Institutes of Health, Bethesda, MD, 20892, USA.

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.
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http://dx.doi.org/10.1186/s12864-020-07241-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7771096PMC
December 2020

Sirolimus augments hematopoietic stem and progenitor cell regeneration following hematopoietic insults.

Stem Cells 2021 Feb 12;39(2):240-252. Epub 2020 Dec 12.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.

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.
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http://dx.doi.org/10.1002/stem.3313DOI Listing
February 2021

Eltrombopag monotherapy can improve hematopoiesis in patients with low to intermediate risk-1 myelodysplastic syndrome.

Haematologica 2020 12 1;105(12):2785-2794. Epub 2020 Dec 1.

National Heart, Lung, and Blood Institute.

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.
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http://dx.doi.org/10.3324/haematol.2020.249995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7716353PMC
December 2020

Comprehensive analysis of single-cell RNA sequencing data from healthy human marrow hematopoietic cells.

BMC Res Notes 2020 Nov 10;13(1):514. Epub 2020 Nov 10.

Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, 20892, USA.

Objective: Single cell methodology enables detection and quantification of transcriptional changes and unravelling dynamic aspects of the transcriptional heterogeneity not accessible using bulk sequencing approaches. We have applied single-cell RNA-sequencing (scRNA-seq) to fresh human bone marrow CD34 cells and profiled 391 single hematopoietic stem/progenitor cells (HSPCs) from healthy donors to characterize lineage- and stage-specific transcription during hematopoiesis.

Results: Cells clustered into six distinct groups, which could be assigned to known HSPC subpopulations based on lineage specific genes. Reconstruction of differentiation trajectories in single cells revealed four committed lineages derived from HSCs, as well as dynamic expression changes underlying cell fate during early erythroid-megakaryocytic, lymphoid, and granulocyte-monocyte differentiation. A similar non-hierarchical pattern of hematopoiesis could be derived from analysis of published single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq), consistent with a sequential relationship between chromatin dynamics and regulation of gene expression during lineage commitment (first, altered chromatin conformation, then mRNA transcription). Computationally, we have reconstructed molecular trajectories connecting HSCs directly to four hematopoietic lineages. Integration of long noncoding RNA (lncRNA) expression from the same cells demonstrated mRNA transcriptome, lncRNA, and the epigenome were highly homologous in their pattern of gene activation and suppression during hematopoietic cell differentiation.
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http://dx.doi.org/10.1186/s13104-020-05357-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7653854PMC
November 2020

Dnmt3a-null hematopoietic stem and progenitor cells expand after busulfan treatment.

Exp Hematol 2020 11 20;91:39-45.e2. Epub 2020 Sep 20.

Section of Infectious Diseases, Department of Pediatrics, Stem Cells and Regenerative Medicine Center, Baylor College of Medicine, Houston, TX. Electronic address:

Mutations in the gene encoding DNA methyltransferase 3A (DNMT3A) comprise the majority of mutations found in clonal hematopoiesis (CH), an age-related condition that was recently found to affect outcomes in patients undergoing hematopoietic stem cell transplant (HSCT). Recent studies have indicated that patients with CH have worse prognoses after HSCT, suggesting stress imposed by HSCT preconditioning agents may impact hematopoietic stem cell (HSC) dynamics in transplant recipients. In this study, we used a competitive transplantation mouse model to investigate how treatment with the common preconditioning agents 5-fluorouracil (5-FU) and busulfan (BU) affect the prevalence of Dnmt3a HSCs and progenitor cells in competition with wild-type cells. We found that, though sufficient to deplete peripheral blood counts, 5-FU preconditioning did not significantly alter the frequency of Dnmt3a-null hematopoietic stem and progenitor cells (HSPCs) in mosaic mice. In contrast, mice treated with BU had a sevenfold decline in total bone marrow cells and an increase in Dnmt3a-null HSPCs that was detectable in peripheral blood. Indeed, even though all mosaic mice had a starting engraftment of ∼10%-40%, 85%-100% of HSPCs were Dnmt3a-null in four of seven mice after BU treatment, indicating these cells expand dramatically during recovery. Overall, these results suggest that individual preconditioning regimens have different effects on the expansion of Dnmt3a-mutant cells in patients with pre-existing CH. Thus, the presence of CH-associated mutants should be evaluated prior to selecting preconditioning regimens for HSCT.
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http://dx.doi.org/10.1016/j.exphem.2020.09.192DOI Listing
November 2020

Conventional Co-Housing Modulates Murine Gut Microbiota and Hematopoietic Gene Expression.

Int J Mol Sci 2020 Aug 26;21(17). Epub 2020 Aug 26.

Laboratory of Cancer Biology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.

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-KitLin) 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-KitSca-1Lin) 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.
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http://dx.doi.org/10.3390/ijms21176143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7503692PMC
August 2020

Deficit of circulating CD19 CD24 CD38 regulatory B cells in severe aplastic anaemia.

Br J Haematol 2020 08 20;190(4):610-617. Epub 2020 Apr 20.

Hematology Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA.

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. CD24 CD38 regulatory B cells (Bregs) suppress CTLs and Th1 responses, and induce Tregs via interleukin 10 (IL-10). We investigated circulating B-cell subpopulations, including CD24 CD38 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. CD24 CD38 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. CD24 CD38 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 CD24 CD38 Bregs, especially in very severe AA, with recovery after IST suggest Breg deficits may contribute to the pathophysiology of immune AA.
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http://dx.doi.org/10.1111/bjh.16651DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496711PMC
August 2020

Attenuation of immune-mediated bone marrow damage in conventionally housed mice.

Mol Carcinog 2020 02 2;59(2):237-245. Epub 2020 Jan 2.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland.

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 β-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.
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http://dx.doi.org/10.1002/mc.23151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7432962PMC
February 2020

Eltrombopag maintains human hematopoietic stem and progenitor cells under inflammatory conditions mediated by IFN-γ.

Blood 2019 05 25;133(19):2043-2055. Epub 2019 Feb 25.

National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD.

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.
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http://dx.doi.org/10.1182/blood-2018-11-884486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509545PMC
May 2019

Long noncoding RNAs of single hematopoietic stem and progenitor cells in healthy and dysplastic human bone marrow.

Haematologica 2019 05 13;104(5):894-906. Epub 2018 Dec 13.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health.

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 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. ().
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http://dx.doi.org/10.3324/haematol.2018.208926DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6518886PMC
May 2019

Epidemiological, clinical and genetic characterization of aplastic anemia patients in Pakistan.

Ann Hematol 2019 Feb 13;98(2):301-312. Epub 2018 Nov 13.

Department of Biochemistry, Quaid-i-Azam University, Islamabad, 44000, Pakistan.

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.
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http://dx.doi.org/10.1007/s00277-018-3542-zDOI Listing
February 2019

Macrophage TNF-α licenses donor T cells in murine bone marrow failure and can be implicated in human aplastic anemia.

Blood 2018 12 25;132(26):2730-2743. Epub 2018 Oct 25.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD.

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.
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http://dx.doi.org/10.1182/blood-2018-05-844928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307988PMC
December 2018

Interleukin-18 plays a dispensable role in murine and likely also human bone marrow failure.

Exp Hematol 2019 01 12;69:54-64.e2. Epub 2018 Oct 12.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.

Interleukin-18 (IL-18), also known as interferon-gamma (IFN-γ)-inducing factor, is involved in Th1 responses and regulation of immunity. Accumulating evidence implicates IL-18 in autoimmune diseases, but little is known of its role in acquired aplastic anemia (AA), the immune-mediated destruction of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs). IL-18 protein levels were significantly elevated in sera of severe AA (SAA) patients, including both responders and nonresponders assayed before treatment, and decreased after treatment. IL-18 receptor (IL-18R) was expressed on HSPCs. Co-culture of human BM CD34 cells from healthy donors with IL-18 upregulated genes in the helper T-cell and Notch signaling pathways and downregulated genes in the cell cycle regulation, telomerase, and IL-6 signaling pathways. Plasma IL-18 levels were also elevated in murine models of immune-mediated BM failure. However, deletion of IL-18 in donor lymph node cells or deletions of either IL-18 or IL-18R in recipients did not attenuate elevations of circulating IFN-γ, tumor necrosis factor-alpha, or IL-6, nor did they alleviate BM failure. In summary, our findings suggest that, although increased circulating IL-18 is a feature of SAA, it may reflect an aberrant immune response but be dispensable to the pathogenesis of AA.
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http://dx.doi.org/10.1016/j.exphem.2018.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309608PMC
January 2019

Aptamer-based proteomics of serum and plasma in acquired aplastic anemia.

Exp Hematol 2018 12 9;68:38-50. Epub 2018 Oct 9.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health (NIH), Bethesda, MD, USA.

Single-stranded oligonucleotides containing deoxyuridine are aptamers (SOMAmers) that can bind proteins with high specificity and affinity and slow dissociation rates. SOMAscan, an aptamer-based proteomic technology, allows measurement of more than 1,300 proteins simultaneously for the identification of new disease biomarkers. The aim of the present study was to identify new serum and plasma protein markers for diagnosis of acquired aplastic anemia (AA) and response to immunosuppressive therapies (IST). SOMAscan was used to screen 1,141 serum proteins in 28 AA patients before and after therapy and 1,317 plasma proteins in seven SAA patients treated with standard IST and a thrombopoietin receptor agonist. From our analysis, 19 serum and 28 plasma proteins were identified as possible candidate diagnostic and prognostic markers. A custom immunobead-based multiplex assay with five selected serum proteins (BMP-10, CCL17, DKK1, HGF, and SELL) was used for validation in a verification set (n = 65) of samples obtained before and after IST and in a blinded validation cohort at baseline (n = 16). After technical validation, four biomarkers were employed to predict diagnosis (accuracy, 88%) and long-term response to IST (accuracy, 79%). In conclusion, SOMAscan is a powerful tool for the identification of new biomarkers. We propose further larger studies to validate new candidate serum and plasma diagnostic and prognostic markers of AA.
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http://dx.doi.org/10.1016/j.exphem.2018.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748047PMC
December 2018

PD-1 deficiency augments bone marrow failure in a minor-histocompatibility antigen mismatch lymphocyte infusion model.

Exp Hematol 2018 06 7;62:17-23. Epub 2018 Mar 7.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health Bethesda, MD, USA.

Although PD-1 blockade has revolutionized cancer immunotherapy, immune-related adverse events (irAEs) present life-threatening complications. Recent reports of aplastic anemia (AA) as irAEs implicate PD-1/PD-L1 as important in preventing immune-mediated destruction of the hematopoietic niche. Infusion of PD-1-deficient (PD-1 knockout [KO]) lymph node (LN) cells into minor-antigen mismatched mice resulted in early mortality, as well as more severe bone marrow (BM) hypoplasia, anemia, and BM microarchitecture disruption in PD-1 KO LN-infused mice relative to mice that received B6 LN cell infusion. Mice that received PD-1 KO LN cells had more CD8 T-cell infiltration of the BM and greater expansion of H60-specific CD8 T cells than did their B6 LN-infused counterparts. In the spleen, CD8 T cells were skewed to an effector memory phenotype, suggesting accelerated differentiation of PD-1 KO T cells. Our data suggest that PD-1 dysregulation has a role in murine BM failure and vigilance in irAE monitoring may be desirable to treat early AA and related cytopenias.
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http://dx.doi.org/10.1016/j.exphem.2018.03.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5962409PMC
June 2018

Deep sequencing and flow cytometric characterization of expanded effector memory CD8CD57 T cells frequently reveals T-cell receptor Vβ oligoclonality and CDR3 homology in acquired aplastic anemia.

Haematologica 2018 05 1;103(5):759-769. Epub 2018 Feb 1.

Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, MD, USA.

Oligoclonal expansion of CD8 CD28 lymphocytes has been considered indirect evidence for a pathogenic immune response in acquired aplastic anemia. A subset of CD8 CD28 cells with CD57 expression, termed effector memory cells, is expanded in several immune-mediated diseases and may have a role in immune surveillance. We hypothesized that effector memory CD8CD28CD57 cells may drive aberrant oligoclonal expansion in aplastic anemia. We found CD8CD57 cells frequently expanded in the blood of aplastic anemia patients, with oligoclonal characteristics by flow cytometric Vβ usage analysis: skewing in 1-5 Vβ families and frequencies of immunodominant clones ranging from 1.98% to 66.5%. Oligoclonal characteristics were also observed in total CD8 cells from aplastic anemia patients with CD8CD57 cell expansion by T-cell receptor deep sequencing, as well as the presence of 1-3 immunodominant clones. Oligoclonality was confirmed by T-cell receptor repertoire deep sequencing of enriched CD8CD57 cells, which also showed decreased diversity compared to total CD4 and CD8 cell pools. From analysis of complementarity-determining region 3 sequences in the CD8 cell pool, a total of 29 sequences were shared between patients and controls, but these sequences were highly expressed in aplastic anemia subjects and also present in their immunodominant clones. In summary, expansion of effector memory CD8 T cells is frequent in aplastic anemia and mirrors Vβ oligoclonal expansion. Flow cytometric Vβ usage analysis combined with deep sequencing technologies allows high resolution characterization of the T-cell receptor repertoire, and might represent a useful tool in the diagnosis and periodic evaluation of aplastic anemia patients. (Registered at ).
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http://dx.doi.org/10.3324/haematol.2017.176701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5927970PMC
May 2018

Heterozygous variants in bone marrow failure and myeloid neoplasms.

Blood Adv 2018 01 4;2(1):36-48. Epub 2018 Jan 4.

Department of Haematological Medicine, King's College Hospital, London, United Kingdom.

Biallelic germline mutations in (regulator of telomere elongation helicase 1) result in pathologic telomere erosion and cause dyskeratosis congenita. However, the role of mutations in other bone marrow failure (BMF) syndromes and myeloid neoplasms, and the contribution of monoallelic mutations to disease development are not well defined. We screened 516 patients for germline mutations in telomere-associated genes by next-generation sequencing in 2 independent cohorts; one constituting unselected patients with idiopathic BMF, unexplained cytopenia, or myeloid neoplasms (n = 457) and a second cohort comprising selected patients on the basis of the suspicion of constitutional/familial BMF (n = 59). Twenty-three variants were identified in 27 unrelated patients from both cohorts: 7 variants were likely pathogenic, 13 were of uncertain significance, and 3 were likely benign. Likely pathogenic variants were identified in 9 unrelated patients (7 heterozygous and 2 biallelic). Most patients were suspected to have constitutional BMF, which included aplastic anemia (AA), unexplained cytopenia, hypoplastic myelodysplastic syndrome, and macrocytosis with hypocellular bone marrow. In the other 18 patients, variants were likely benign or of uncertain significance. Telomeres were short in 21 patients (78%), and 3' telomeric overhangs were significantly eroded in 4. In summary, heterozygous variants were associated with marrow failure, and telomere length measurement alone may not identify patients with telomere dysfunction carrying variants. Pathogenicity assessment of heterozygous variants relied on a combination of clinical, computational, and functional data required to avoid misinterpretation of common variants.
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http://dx.doi.org/10.1182/bloodadvances.2017008110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761623PMC
January 2018

Immune thrombocytopenia is associated with persistently deranged fibrosis-related seromarker profiles but low bone marrow fibrosis grades: A 2-year observational study on thrombopoietin receptor agonist treatment.

Platelets 2019 2;30(2):222-228. Epub 2018 Jan 2.

d Department of Pediatrics, Division of Hematology/Oncology , Weill Cornell Medical College , New York, NY , USA.

Bone marrow (BM) fibrosis is a potential side effect of thrombopoietin receptor agonist (TPO-RA) treatment. We aimed to investigate stromal seromarker profiles and growth factors in order to elucidate pathogenic and dynamic aspects of immune thrombocytopenia (ITP)-related BM fibrosis before and during TPO-RA treatment. Connective tissue metabolites [procollagen I and III peptides (PINP/PIIINP); hyaluronan (HYA), C-terminal-telopeptide (ICTP), and fibrosis-related growth factors (transforming growth factor-beta (TGF-beta), HGF, basic fibroblast growth factor)] were measured in blood samples acquired before initiation of TPO-RA and subsequently at 6-month intervals for up to 2 years. BM fibrosis was graded MF-0 in 8 (18%), MF-1 30 (65%), and MF-2 8 (18%) in the last available BM biopsy. In the 21 patients having more than one biopsy, the grade of fibrosis from the first to the last available biopsy decreased in 2 (10%), remained unchanged in 15 (71%), and increased in 4 (19%). Pretreatment levels of PIIINP, PINP, ICTP, and HYA were significantly increased in ITP versus controls. PINP, PIIINP, and HYA decreased on TPO-RA; ICTP remained unchanged. PINP:ICTP was lower before and during treatment compared to controls. Pretreatment, TGF-beta was lower than in controls; HGF exhibited the opposite pattern. HYA, ICTP, and TGF-beta tended to increase while PINP and platelet-derived growth factor tended to decrease with increasing fibrosis grade. In conclusion, ITP is associated with deranged patterns of extracellular matrix seromarkers and growth factors, indicating that BM stromal remodeling is enhanced. During TPO-RA treatment for up to 2 years, this profile was partially reversed while mild BM reticulin fibrosis was still present in the majority of patients. These observations likely reflect a BM injury by autoimmunity that is modified by TPO-RA.
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http://dx.doi.org/10.1080/09537104.2017.1411586DOI Listing
April 2019

Single-cell RNA-seq reveals a distinct transcriptome signature of aneuploid hematopoietic cells.

Blood 2017 12 13;130(25):2762-2773. Epub 2017 Oct 13.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD.

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.
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http://dx.doi.org/10.1182/blood-2017-08-803353DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5746166PMC
December 2017

Persistent elevation of plasma thrombopoietin levels after treatment in severe aplastic anemia.

Exp Hematol 2018 02 20;58:39-43. Epub 2017 Sep 20.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD.

Although hematopoietic growth factors are found at high levels in aplastic anemia (AA) patients, little is known about their dynamic change over time after treatment. We examined plasma concentrations of hematopoietic growth factors sequentially in 55 severe AA patients, including 45 treatment-naive patients who had received immunosuppressive therapy (IST) or IST and eltrombopag, and 10 IST-refractory patients who had received eltrombopag only, focusing on thrombopoietin (TPO). TPO concentrations were much higher than normal in patients before treatment and then decreased in responders but not in nonresponders. We followed up on a cohort of nine patients who obtained stable complete remission for up to 7 years after IST and found that TPO levels declined gradually by 3 months after treatment, accompanying an increase in platelet counts, but stabilized at levels higher than normal. An inverse correlation was noted between TPO levels and platelet counts. The increased plasma TPO levels could be required to maintain normal platelet counts in remission and could also be attributed to reduced consumption by circulating platelets.
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http://dx.doi.org/10.1016/j.exphem.2017.09.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5815891PMC
February 2018

Telomerase enzyme deficiency promotes metabolic dysfunction in murine hepatocytes upon dietary stress.

Liver Int 2018 01 19;38(1):144-154. Epub 2017 Aug 19.

Department of Internal Medicine, Ribeirão Preto School of Medicine, University of São Paulo, Ribeirão Preto, SP, Brazil.

Background & Aims: Short telomeres and genetic telomerase defects are risk factors for some human liver diseases, ranging from non-alcoholic fatty liver disease and non-alcoholic steatohepatitis to cirrhosis. In murine models, telomere dysfunction has been shown to metabolically compromise hematopoietic cells, liver and heart via the activation of the p53-PGC axis.

Methods: Tert- and Terc-deficient mice were challenged with liquid high-fat diet. Liver metabolic contents were analysed by CE-TOFMS and liver fat content was confirmed by confocal and electronic microscopy.

Results: Tert-deficient but not Terc-deficient mice develop hepatocyte injury and frank steatosis when challenged with liquid high-fat diet. Upon high-fat diet, Tert hepatocytes fail to engage the citric acid cycle (TCA), with an imbalance of NADPH/NADP and NADH/NAD ratios and depletion of intermediates of TCA cycle, such as cis-aconitic acid. Telomerase deficiency caused an intrinsic metabolic defect unresponsive to environmental challenge. Chemical inhibition of telomerase by zidovudine recapitulated the abnormal Tert metabolic phenotype in Terc hepatocytes.

Conclusions: Our findings indicate that in telomeropathies short telomeres are not the only molecular trigger and telomerase enzyme deficiency provokes hepatocyte metabolic dysfunction, abrogates response to environmental challenge, and causes cellular injury and steatosis, providing a mechanism for liver damage in telomere diseases.
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http://dx.doi.org/10.1111/liv.13529DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5741503PMC
January 2018

Rapamycin is highly effective in murine models of immune-mediated bone marrow failure.

Haematologica 2017 10 20;102(10):1691-1703. Epub 2017 Jul 20.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.

Acquired aplastic anemia, the prototypical bone marrow failure disease, is characterized by pancytopenia and marrow hypoplasia. Most aplastic anemia patients respond to immunosuppressive therapy, usually with anti-thymocyte globulin and cyclosporine, but some relapse on cyclosporine withdrawal or require long-term administration of cyclosporine to maintain blood counts. In this study, we tested efficacy of rapamycin as a new or alternative treatment in mouse models of immune-mediated bone marrow failure. Rapamycin ameliorated pancytopenia, improved bone marrow cellularity, and extended animal survival in a manner comparable to the standard dose of cyclosporine. Rapamycin effectively reduced Th1 inflammatory cytokines interferon-γ and tumor necrosis factor-α, increased the Th2 cytokine interleukin-10, stimulated expansion of functional regulatory T cells, eliminated effector CD8 T cells (notably T cells specific to target cells bearing minor histocompatibility antigen H60), and preserved hematopoietic stem and progenitor cells. Rapamycin, but not cyclosporine, reduced the proportion of memory and effector T cells and maintained a pool of naïve T cells. Cyclosporine increased cytoplasmic nuclear factor of activated T-cells-1 following T-cell receptor stimulation, whereas rapamycin suppressed phosphorylation of two key signaling molecules in the mammalian target of rapamycin pathway, S6 kinase and protein kinase B. In summary, rapamycin was an effective therapy in mouse models of immune-mediated bone marrow failure, acting through different mechanisms to cyclosporine. Its specific expansion of regulatory T cells and elimination of clonogenic CD8 effectors support its potential clinical utility in the treatment of aplastic anemia.
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http://dx.doi.org/10.3324/haematol.2017.163675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5622853PMC
October 2017

Optimization and standardization of fluorescent cell barcoding for multiplexed flow cytometric phenotyping.

Cytometry A 2017 07 10;91(7):694-703. Epub 2017 Jul 10.

Center for Human Immunology, Autoimmunity, and Inflammation, NIH, Bethesda, Maryland, 20892-1202.

Fluorescent cell barcoding (FCB) is a cell-based multiplexing technique for high-throughput flow cytometry. Barcoded samples can be stained and acquired collectively, minimizing staining variability and antibody consumption, and decreasing required sample volumes. Combined with functional measurements, FCB can be used for drug screening, signaling profiling, and cytokine detection, but technical issues are present. We optimized the FCB technique for routine utilization using DyLight 350, DyLight 800, Pacific Orange, and CBD500 for barcoding six, nine, or 36 human peripheral blood specimens. Working concentrations of FCB dyes ranging from 0 to 500 μg/ml were tested, and viability dye staining was optimized to increase robustness of data. A five-color staining with surface markers for Vβ usage analysis in CD4 and CD8 T cells was achieved in combination with nine sample barcoding. We provide improvements of the FCB technique that should be useful for multiplex drug screening and for lymphocyte characterization and perturbations in the diagnosis and during the course of disease. Published 2017 by Wiley Periodicals, Inc., on behalf of International Society for Advancement of Cytometry. This article is a US government work and as such, is in the public domain in the United States of America.
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http://dx.doi.org/10.1002/cyto.a.23162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5612408PMC
July 2017

T Cell Transcriptomes from Paroxysmal Nocturnal Hemoglobinuria Patients Reveal Novel Signaling Pathways.

J Immunol 2017 07 19;199(2):477-488. Epub 2017 Jun 19.

Cell Biology Section, Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892; and.

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 () 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 (, , , , , , , , and ) 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.
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http://dx.doi.org/10.4049/jimmunol.1601299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543699PMC
July 2017

Hematopoietic Aging Biomarkers in Mice.

J Aging Sci 2017 Apr 6;5(1). Epub 2017 Feb 6.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD USA.

We analyzed hematopoietic phenotypes in (PL) mice at young (2-9 months), middle (22-23 months) and old (33-46 months) ages aimed at characterizing age-associated changes in this unique rodent species. We found a significantly higher number of monocytes in old PL mice in peripheral blood, and higher proportions of CD44 cells in blood, spleen and bone marrow in old PL mice than in middle and young counterparts. We conclude that elevated blood monocyte counts and up-regulated hematopoietic cell CD44 expression are two useful aging biomarkers for PL mice.
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http://dx.doi.org/10.4172/2329-8847.1000169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5469418PMC
April 2017

Eltrombopag Added to Standard Immunosuppression for Aplastic Anemia.

N Engl J Med 2017 04;376(16):1540-1550

From the Hematology Branch (D.M.T., T.W., R.D., B.D., O.R., B.W., J.V., J.L., X.F., M.D., H.L., A.L., C.E.D., N.S.Y.) and the Office of Biostatistics Research (C.W.), National Heart, Lung, and Blood Institute, and the Nursing Research and Translational Science Section, Department of Nursing (M.B.), and the Hematology Section, Department of Laboratory Medicine (K.R.C.), Clinical Center - all at the National Institutes of Health, Bethesda, MD; and the Division of Clinical Hematology, Antônio Ermírio de Moraes Cancer Center, Hospital A Beneficência Portuguesa de São Paulo, São Paulo (P.S.).

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 .).
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http://dx.doi.org/10.1056/NEJMoa1613878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5548296PMC
April 2017

Whole transcriptome sequencing identifies increased CXCR2 expression in PNH granulocytes.

Br J Haematol 2017 04 1;177(1):136-141. Epub 2017 Feb 1.

Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, MD, USA.

The aetiology of paroxysmal nocturnal haemoglobinuria (PNH) is a somatic mutation in the X-linked phosphatidylinositol glycan class A gene (PIGA), resulting in global deficiency of glycosyl phosphatidylinositol-anchored proteins (GPI-APs). This study applied RNA-sequencing to examine functional effects of the PIGA mutation in human granulocytes. CXCR2 expression was increased in GPI-AP compared to GPI-AP granulocytes. Macrophage migration inhibitory factor, a CXCR2 agonist, was significantly higher in plasma of PNH patients. Nuclear factor-κB phosphorylation was upregulated in GPI-AP compared with GPI-AP granulocytes. Our data suggest novel mechanisms in PNH, not obviously predicted by decreased production of the GPI moiety.
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http://dx.doi.org/10.1111/bjh.14502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5378616PMC
April 2017

Effect of thrombopoietin-receptor agonists on circulating cytokine and chemokine levels in patients with primary immune thrombocytopenia (ITP).

Platelets 2017 Jul 7;28(5):478-483. Epub 2016 Nov 7.

e Department of Paediatric Haematology/Oncology , Weill Cornell Medical College , New York , NY , USA.

Background: Thrombopoietin-receptor-agonists (TPO-RAs) increase platelet production in Immune Thrombocytopenia (ITP) by stimulating Mpl. The effect of TPO-RAs on inflammatory cytokine production in ITP patients has not been well investigated.

Methods: Plasma samples from 48 ITP patients treated with TPO-RAs (median age 50 years (inter-quartile range; IQR 20-69), median platelet counts 24 × 10/L (IQR 15-47 × 10/L), 28 females) and 16 healthy controls (nine females, median age 37 years, IQR 22-51 years) were collected before and during treatment, and analyzed for a panel of cytokines and chemokines by enzyme-linked immunosorbent assay and immuno-bead-based multiplex assay.

Results: Elevated levels of C-X-C motif chemokine 10 (CXCL10; p < 0.001) and osteoprotegerin (OPG; p < 0.05) were observed in pretreatment samples compared to controls; these levels decreased during 6 months of treatment. Pretreatment levels of transforming growth factor (TGF)-β were lower than in healthy controls and increased after 6 months of treatment (p < 0.05). Levels of sCD40L increased after 6 months of treatment (p < 0.05), but decreased thereafter to pretreatment values. The increase in TGF-β and sCD40L may reflect increased platelet turnover. Levels of tumor necrosis factor (TNF)-α, interferon (IFN)-γ and interleukin (IL)-10 did not change during treatment.

Conclusion: These findings suggest that treatment with TPO-RA creates a more balanced steady-state of immune activation.
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http://dx.doi.org/10.1080/09537104.2016.1235691DOI Listing
July 2017

Epigenetic landscape of the TERT promoter: a potential biomarker for high risk AML/MDS.

Br J Haematol 2016 Nov 19;175(3):427-439. Epub 2016 Jul 19.

Hematology Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA.

Although recent observations implicate the importance of telomerase activity in acute myeloid leukaemia (AML), the roles of epigenetic regulations of the TERT gene in leukaemogenesis, drug resistance and clinical prognosis in AML are not fully understood. We developed a quantitative pyrosequencing-based methylation assay covering the TERT proximal promoter and a partial exon 1 (TERTpro/Ex1) region and tested both cell lines and primary leukaemia cells derived from AML and AML with preceding myelodysplastic syndrome (AML/MDS) patients (n = 43). Prognostic impact of methylation status of the upstream TERT promoter region was assessed by the Kaplan-Meier method. The activity of the telomerase inhibitor, imetelstat, was measured using leukaemia cell lines. The TERTpro/Ex1 region was highly methylated in all cell lines and primary leukaemia cells showed diverse methylation profiles. Most cases showed hypermethylated regions at the upstream TERTpro/Ex1 region, which were associated with inferior patient survival. TERTpro/Ex1 methylation status was correlated with the cytotoxicity to imetelstat and its combination with hypomethylating agent enhanced the cytotoxicity of imetelstat. AML cell lines and primary blasts harbour distinct TERTpro/Ex1 methylation profiles that could serve as a prognostic biomarker of AML. However, validation in a large cohort of patients is necessary to confirm our findings.
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http://dx.doi.org/10.1111/bjh.14244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5245983PMC
November 2016

A plasma microRNA signature as a biomarker for acquired aplastic anemia.

Haematologica 2017 01 22;102(1):69-78. Epub 2016 Sep 22.

Hematology Branch, National Heart, Lung, and Blood Institute (NHLBI), NIH, Bethesda, MA, USA.

Aplastic anemia is an acquired bone marrow failure characterized by marrow hypoplasia, a paucity of hematopoietic stem and progenitor cells, and pancytopenia of the peripheral blood, due to immune attack on the bone marrow. In aplastic anemia, a major challenge is to develop immune biomarkers to monitor the disease. We measured circulating microRNAs in plasma samples of aplastic anemia patients in order to identify disease-specific microRNAs. A total of 179 microRNAs were analyzed in 35 plasma samples from 13 aplastic anemia patients, 11 myelodysplastic syndrome patients, and 11 healthy controls using the Serum/Plasma Focus microRNA Polymerase Chain Reaction Panel. Subsequently, 19 microRNAs from the discovery set were investigated in the 108 plasma samples from 41 aplastic anemia patients, 24 myelodysplastic syndrome patients, and 43 healthy controls for validation, confirming that 3 microRNAs could be validated as dysregulated (>1.5-fold change) in aplastic anemia, compared to healthy controls. MiR-150-5p (induction of T-cell differentiation) and miR-146b-5p (involvement in the feedback regulation of innate immune response) were elevated in aplastic anemia plasma, whereas miR-1 was decreased in aplastic anemia. By receiver operating characteristic curve analysis, we developed a logistic model with these 3 microRNAs that enabled us to predict the probability of a diagnosis of aplastic anemia with an area under the curve of 0.86. Dysregulated expression levels of the microRNAs became normal after immunosuppressive therapy at 6 months. Specifically, miR-150-5p expression was significantly reduced after successful immunosuppressive therapy, but did not change in non-responders. We propose 3 novel plasma biomarkers in aplastic anemia, in which miR-150-5p, miR-146b-5p, and miR-1 can serve for diagnosis and miR-150-5p for disease monitoring. Clinicaltrials.gov identifiers:00260689, 00217594, 00961064.
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http://dx.doi.org/10.3324/haematol.2016.151076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210234PMC
January 2017