Marrow Erythroid Cells Research Articles

CD59-deficient bone marrow erythroid cells from rats treated with procarbazine and propyl-nitrosourea have mutations in the Pig-a gene.

Procarbazine (PCZ) and N-propyl-N-nitrosourea (PNU) are rodent mutagens and carcinogens. Both induce GPI-anchored marker-deficient mutant-phenotype red blood cells (RBCs) in the flow cytometry-based rat RBC Pig-a assay. In the present study, we traced the origin of the RBC mutant phenotype by analyzing Pig-a mutations in the precursors of RBCs, bone marrow erythroid cells (BMEs). Rats were exposed to a total of 450 mg/kg PCZ hydrochloride or 300 mg/kg PNU, and bone marrow was collected 2, 7, and 10 weeks later. Using a flow cell sorter, we isolated CD59-deficient mutant-phenotype BMEs from PCZ- and PNU-treated rats and examined their endogenous X-linked Pig-a gene by next generation sequencing. Pig-a mutations consistent with the properties of PCZ and PNU were found in sorted mutant-phenotype BMEs. PCZ induced mainly A > T transversions with the mutated A on the nontranscribed strand of the Pig-a gene, while PNU induced mainly T > A transversions with the mutated T on the nontranscribed strand. The treatment-induced mutations were distributed across the protein coding sequence of the Pig-a gene. The causal relationship between BMEs and RBCs and the agent-specific mutational spectra in CD59-deicient BMEs indicate that the rat RBC Pig-a assay, scoring CD59-deficient mutant-phenotype RBCs in peripheral blood, detects Pig-a gene mutation.

A Novel Choice to Correct Inflammation-Induced Anemia in CKD: Oral Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor Roxadustat.

Anemia is a complication of chronic kidney disease (CKD), primarily due to insufficient secretion of erythropoietin (EPO) by the kidney. Erythropoiesis-stimulating agents (ESAs) are used to treat anemia associated with chronic kidney disease. A poor response to ESAs has been associated with inflammation. Inflammation can affect erythrocytes and its production in many ways, but mainly through the inflammatory cytokine IL-6 to stimulate the synthesis of hepcidin in the liver. Hepcidin causes iron insufficiency, which causes erythrocytes to fail to mature normally. In addition, inhibition of bone marrow erythroid precursor cells by inflammatory cytokines such as IL-1 and TNF-α also affects bone marrow hematopoiesis. These cytokines are also important factors leading to EPO resistance. Roxadustat is a new drug for the treatment of renal anemia. In addition to promoting the production of EPO, clinical trials have shown that it can significantly reduce hepcidin and can potentially be used for the treatment of inflammation-induced anemia in CKD.

Erythropoietin-Induced Changes in Bone and Bone Marrow in Mouse Models of Diet-Induced Obesity

Obesity remodels bone and increases bone marrow adipocytes (BMAT), which negatively regulate hematopoiesis and bone. Reduced BMAT could restore altered hematopoiesis and bone features. We analyzed the potential of erythropoietin (EPO), the cytokine required for erythropoiesis, to inhibit BMAT in C57BL6/J mice fed four weeks of a high-fat diet (HFD). Acute EPO administration markedly decreased BMAT in regular chow diet (RCD) and HFD-fed mice, without affecting whole body fat mass. Micro-CT analysis showed EPO reduced trabecular bone in RCD- and HFD-fed mice, but EPO-treated HFD-fed mice maintained cortical bone mineral density and cortical bone volume, which was reduced on RCD. Despite achieving similar increased hematocrits with BMAT loss in RCD- and HFD-fed mice treated with EPO, decreased bone marrow cellularity was only observed in RCD-fed mice concomitant with an increasing percentage of bone marrow erythroid cells. In contrast, in HFD-fed mice, EPO increased endothelial cells and stromal progenitors with a trend toward the normalization of marrow homeostasis. EPO administration increased c-terminal FGF23 and intact serum FGF23 only in HFD-fed mice. These data demonstrate the distinct EPO responses of bone and marrow in normal and obese states, accompanying EPO-induced loss of BMAT.

Dominant Nucleolus in the Progenitor Cell Using Human Bone Marrow Erythroid and Granulocytic Cell Lineages as a Model. A Morphological and Cytochemical Note

Progenitor cells of the human erythroid and granulocytic cell lineages are characterized by the presence of several nucleoli. One of these nucleoli is larger and possesses more fibrillar centres than others. Such nucleolus is apparently dominant in respect of both size and main nucleolar function such as nucleolar-ribosomal RNA transcription. Such nucleolus is also visible in specimens using conventional visualization procedures, in contrast to smaller nucleoli. In the terminal differentiation nucleated stages of the erythroid and granulocytic development, dominant nucleoli apparently disappeared, since these cells mostly contained very small nucleoli of a similar size with one fibrillar centre. Thus, the easily visible dominant nucleoli appear to be useful markers of the progenitor cell state, such as proliferation, and differentiation potential.

Pig-a mutations in bone marrow erythroblasts of rats treated with 7,12-dimethyl-benz[a]anthracene

Flow cytometry-based phenotypic detection of red blood cells (RBCs) deficient in surface markers anchored by glycosylphosphatidylinositol (GPI) is an efficient tool for monitoring somatic mutation in mammalian species. Biochemical considerations suggest that GPI-anchored marker-deficient RBCs found in peripheral blood are due to mutations in the endogenous X-linked phosphatidylinositolglycan, class A gene (Pig-a gene). Yet the linkage between the detected mutant phenotype and the actual mutation in the Pig-a gene is difficult to establish directly in mammalian RBCs that are naturally free of genomic DNA and may have only traces of heavily degraded mRNA. We have traced the origin of the marker-deficient RBC phenotype in the precursors of peripheral RBCs, bone marrow erythroid cells (BMEs, also known as erythroblasts), in rats treated by gavage with 75 mg/kg of the potent mutagen, 7,12-dimethyl-benz[a]anthracene (DMBA). The frequencies of marker-deficient BMEs were significantly increased in DMBA-treated rats. We identified Pig-a mutations in sorted mutant phenotype BMEs. The spectrum of DMBA-induced Pig-a mutations in erythroid lineage cells was identical to the spectra of mutations previously determined for the Pig-a and for another X-linked reporter gene, hypoxanthine-guanine phosphoribosyltransferase gene, in cells of lymphoid lineage, spleen T-lymphocytes. Our observations lend additional support to the hypothesis that GPI-anchored marker-deficient RBCs are true Pig-a mutants.

Upregulation of Pro-inflammatory Cytokine Genes by Parvovirus B19 in Human Bone Marrow Mesenchymal Stem Cells.

Chronic inflammation plays a prominent role in cancer initiation and development. On the other hand, the Inflammation can be established by a number of factors such as viral infections. Parvovirus B19 (B19V) is a pathogen with widespread infection, which infects bone marrow erythroid progenitor cells. It has been shown that B19V can also enter human bone marrow mesenchymal stem cells (BM-MSCs). In this study, we hypothesized that BM-MSCs as the main cellular component of bone marrow niche may be induced to secret pro-inflammatory cytokines after B19V infection. BM-MSCs were cultured up to passage 3. The cells were then subjected to nucleofection to transfer a plasmid containing B19V genome. After 36h, total RNA was extracted and the expression levels of IL-1β, IL-6, TNF-α and NF-κB genes were examined using qRT-PCR. Data analysis showed the significant increase in expression levels of all studied genes in the B19V-transfected cells (P < 0.05). Although further researches are required, our findings for the first time suggest the importance of B19V infection to establish an inflammatory microenvironment in the bone marrow and its involvement in inflammation-related diseases. Finally, based on our results, molecular assay to diagnose B19V infection of BM-MSCs prior to stem cell therapy is strongly recommended.

Single-cell analyses demonstrate that a heme–GATA1 feedback loop regulates red cell differentiation

AbstractErythropoiesis is the complex, dynamic, and tightly regulated process that generates all mature red blood cells. To understand this process, we mapped the developmental trajectories of progenitors from wild-type, erythropoietin-treated, and Flvcr1-deleted mice at single-cell resolution. Importantly, we linked the quantity of each cell's surface proteins to its total transcriptome, which is a novel method. Deletion of Flvcr1 results in high levels of intracellular heme, allowing us to identify heme-regulated circuitry. Our studies demonstrate that in early erythroid cells (CD71+Ter119neg-lo), heme increases ribosomal protein transcripts, suggesting that heme, in addition to upregulating globin transcription and translation, guarantees ample ribosomes for globin synthesis. In later erythroid cells (CD71+Ter119lo-hi), heme decreases GATA1, GATA1-target gene, and mitotic spindle gene expression. These changes occur quickly. For example, in confirmatory studies using human marrow erythroid cells, ribosomal protein transcripts and proteins increase, and GATA1 transcript and protein decrease, within 15 to 30 minutes of amplifying endogenous heme synthesis with aminolevulinic acid. Because GATA1 initiates heme synthesis, GATA1 and heme together direct red cell maturation, and heme stops GATA1 synthesis, our observations reveal a GATA1–heme autoregulatory loop and implicate GATA1 and heme as the comaster regulators of the normal erythroid differentiation program. In addition, as excessive heme could amplify ribosomal protein imbalance, prematurely lower GATA1, and impede mitosis, these data may help explain the ineffective (early termination of) erythropoiesis in Diamond Blackfan anemia and del(5q) myelodysplasia, disorders with excessive heme in colony-forming unit-erythroid/proerythroblasts, explain why these anemias are macrocytic, and show why children with GATA1 mutations have DBA-like clinical phenotypes.

Analysis of mutation in the rat Pig‐a assay: I) studies with bone marrow erythroid cells

We have established a flow cytometry-based Pig-a assay for rat bone marrow erythroid cells (BMEs). The BME Pig-a assay uses a DNA-specific stain and two antibodies: one against the transmembrane transferrin receptor (CD71 marker) and the other against the GPI-anchored complement inhibitory protein (CD59 marker). In F344 male rats treated acutely with a total of 120 mg/kg of N-ethyl-N-nitrosourea (ENU) the frequency of CD59-deficient phenotypically mutant BMEs increased approximately 24-fold compared to the rats concurrently treated with the vehicle. Such an increase of mutant BMEs coincides with increases of CD59-deficient reticulocytes measured in rats treated with similar doses of ENU. Sequence analysis of the endogenous X-linked Pig-a gene of CD59-deficient BMEs revealed that they are Pig-a mutants. The spectrum of ENU-induced Pig-a mutations in these BMEs was consistent with the in vivo mutagenic signature of ENU: 73% of mutations occurred at A:T basepairs, with the mutated T on the nontranscribed strand of the gene. T→A transversion was the most frequent mutation followed by T→C transition; no deletion or insertion mutations were present in the spectrum. Since BMEs are precursors of peripheral red blood cells, our findings suggest that CD59-deficient erythrocytes measured in the flow cytometric erythrocyte Pig-a assay develop from BMEs containing mutations in the Pig-a gene. Thus, the erythrocyte Pig-a assay detects mutation in the Pig-a gene. Environ. Mol. Mutagen. 59:722-732, 2018. © 2018 Wiley Periodicals, Inc.

Thermal injury of the skin induces G-CSF-dependent attenuation of EPO-mediated STAT signaling and erythroid differentiation arrest in mice

Inflammation-mediated impairment of erythropoiesis plays a central role in the development of the anemia of critical illness (ACI). ACI develops despite elevation of endogenous erythropoietin (EPO), does not respond to exogenous erythropoietin (EPO) supplementation, and contributes significantly to transfusion requirements in burned patients. We have reported previously that the reduction of red blood cell mass in the bone marrow of a burn-injured ACI mouse model is granulocyte colony-stimulating factor (G-CSF) dependent. Given that elevated G-CSF levels also have been associated with lower hemoglobin levels and increased transfusion requirements in trauma victims, we postulated that G-CSF mediates postburn EPO resistance. In ACI mice, we found that bone marrow erythroid differentiation, viability, and proliferation are impaired after thermal injury of the skin. These changes in the marrow were associated with attenuated phosphorylation of known EPO-responsive signaling nodes, signal transducer and activator of transcription 5 (STAT5) Y694 and STAT3 S727, in bone marrow erythroid cells and developed despite highly elevated levels of endogenous EPO. Severely blunted STAT5 Y694 phosphorylation in bone marrow erythroid cells after exogenous EPO supplementation confirmed that EPO signaling was impaired in ACI mice. Importantly, parenteral administration of anti-G-CSF largely rescued postburn bone marrow erythroid differentiation arrest and EPO signaling in erythroid cells. Together, these data provide strong evidence for a role for G-CSF in the development of ACI after burn injury through suppression of EPO signaling in bone marrow erythroid cells.

Persistent injury-associated anemia: the role of the bone marrow microenvironment

BackgroundThe regulation of erythropoiesis involves hematopoietic progenitor cells, bone marrow stroma, and the microenvironment. Following severe injury, a hypercatecholamine state develops that is associated with increased mobilization of hematopoietic progenitor cells to peripheral blood and decreased growth of bone marrow erythroid progenitor cells that manifests clinically as a persistent injury-associated anemia. Changes within the bone marrow microenvironment influence the development of erythroid progenitor cells. Therefore, we sought to determine the effects of lung contusion, hemorrhagic shock, and chronic stress on the hematopoietic cytokine response. Materials and methodsBone marrow was obtained from male Sprague–Dawley rats (n = 6/group) killed 7 d after lung contusion followed by hemorrhagic shock (LCHS) or LCHS followed by daily chronic restraint stress (LCHS/CS). End point polymerase chain reaction was performed for interleukin-1β, interleukin-10, stem cell factor, transforming growth factor-β, high-mobility group box-1 (HMGB-1), and B-cell lymphoma-extra large. ResultsSeven days following LCHS and LCHS/CS, bone marrow expression of prohematopoietic cytokines (interleukin-1β, interleukin-10, stem cell factor, and transforming growth factor-β) was significantly decreased, and bone marrow expression of HMGB-1 was significantly increased. B-cell lymphoma-extra large bone marrow expression was not affected by LCHS or LCHS/CS (naïve: 44 ± 12, LCHS: 44 ± 12, LCHS/CS: 37 ± 1, all P > 0.05). ConclusionsThe bone marrow microenvironment was significantly altered following severe trauma in a rodent model. Prohematopoietic cytokines were downregulated, and the proinflammatory cytokine HMGB-1 had increased bone marrow expression. Modulation of the bone marrow microenvironment may represent a therapeutic strategy following severe trauma to alleviate persistent injury-associated anemia.

Revisiting erythroleukemia.

The 2016 WHO classification of hematopoietic and lymphoid neoplasms alters the diagnostic criteria for erythroleukemia, including eliminating the erythroid/myeloid type of acute erythroleukemia, which was a prior subcategory of acute myeloid leukemia, not otherwise specified. Only pure erythroid leukemia remains in the WHO classification. This review will summarize the literature that contributed to that classification change as well as recent literature on the significance of the change. There is now a large body of literature on the negative prognostic impact of erythroid predominance, defined as 50% or more bone marrow erythroid cells, in myelodysplastic syndromes (MDSs). Recent studies have found similarities between erythroleukemia, especially the erythroid/myeloid type, and the erythroid-rich MDS cases. On the basis of these data, the WHO now reclassifies cases of the prior erythroid/myeloid acute erythroleukemia group based on the total blast cell count. This change moves such cases into an MDS category, usually MDS with excess blasts. This approach, however, may cloud the significance of erythroid predominance in this group of patients. The report clarifies the current criteria for a diagnosis of erythroleukemia as well as the ongoing challenges in classifying this group of erythroid rich bone marrow disorders.

Histological features of bone marrow in paediatric patients during the asymptomatic phase of early-stage Black African sickle cell anaemia

Bone marrow histological features of sickle cell anaemia (SCA) patients during early stages and in the asymptomatic phase of the disease appear an interesting area of study, representing early-stage consequences of SCA with a close relation to its pathophysiology. Unfortunately, this field of research has never been specifically addressed before. Bone marrow biopsies from 26 consecutive Black African SCA patients (M:F=1.6:1; age 2-17 years), free of clinical signs of chronic bone marrow damage, with no recent history of symptomatic vaso-occlusive episodes, and waiting for haematopoietic stem cell transplantation (HSCT), underwent morphological, immunohistochemical and electron microscopy evaluation. Additional comparison with three bone marrow specimens from post-HSCT SCA patients and 10 bone marrow specimens from AS healthy carriers was performed. Bone marrow of SCA patients was normocellular or slighly hypercellular in all cases. Erythroid hyperplasia was a common feature. Myeloid lineage was slightly decreased with normal to slightly diminished neutrophilic granulocytes; CD68 positive monocytic-macrophagic cells appeared slightly increased, with a predominant CD163 positive M2/M(Hb) phenotype. A positive correlation was found between haemoglobin values and number of bone marrow erythroid cells (R2=0.15, p=0.05). Intravascular and interstitial clusters of erythroid sickle cells were found in bone marrow of pre-HSCT homozygous SS SCA patients, as well as heterozygous AS healthy carriers, and the single post-HSCT patient matched to an AS health carrier donor.

The Interplay of GATA1 with Heme Regulates an Erythroid Cell's Differentiation

GATA1 promotes the transcription of ALAS2, the first and rate limiting step of heme synthesis, and the transcription of many other erythroid-specific genes. It also increases its own transcription while silencing proliferation genes active in early progenitors and thus assures that erythroid differentiation correctly initiates. Heme then transcriptionally and translationally upregulates globin to guarantee adequate hemoglobin production in each cell as it matures. In mice lacking the heme exporter, FLVCR1, excess heme and ROS accumulate and erythropoiesis fails at the CFU-E/proerythroblast stage, resulting in a severe macrocytic anemia (HGB 4.4±0.97 vs 14.8±0.57 g/dL; MCV 66.9±6.2 vs 48.4±0.65 fL). To determine how excess heme causes ineffective erythropoiesis and whether heme is key to terminating differentiation in normal erythroid cells, we performed RNA sequencing of single early erythroid cells (BFU-E to basophilic erythroblasts) from wildtype control and Flvcr1-deleted mice and linked this transcription data to the total quantity of Ter119 on that cell. Principal component analysis (PCA) identified 4 transcriptionally unique clusters A, B, C, & D, which contained cells with negative, low, intermediate, and high Ter119 levels respectively. α- and β-globin transcription were highly correlated (r=0.975), occurred in all cells, increased as Ter119 expression increased, and upregulated in Flvcr1-deleted cells. Gene set enrichment analysis (GSEA) comparing control cells to Flvcr1-deleted cells revealed excess heme results in significant downregulation of the hallmark heme metabolism pathway genes (heme biosynthesis and erythroid differentiation genes), upregulation of the ribosome pathway genes, and no alteration of the P53 pathway genes. All eight heme biosynthetic enzyme genes were expressed equivalently in cluster A cells from control and Flvcr1-deleted mice; however expression in Flvcr1-deleted cells was significantly reduced in clusters B-D. Of the 181 erythroid differentiation genes in the hallmark heme pathway, Gata1 had the greatest reduction (67%) in Flvcr1-deleted cells. Coupled two-way clustering analysis (CTWC) identified 150 genes co-regulated with Gata1 including 106 known GATA1 target genes which were all poorly upregulated in Flvcr1-deleted cells in clusters B-D. Independent microarray analysis of mRNA from control and Flvcr1-deleted CD71+ erythroid cells confirmed low Gata1 mRNA and low GATA1-dependent gene expression in the Flvcr1-deleted cells. To determine if excess heme was directly responsible for Gata1 downregulation, we treated K562, HEL-R, and primary human erythroid marrow cells with aminolevulinic acid (ALA) and iron to increase endogenous heme synthesis. In the primary cells, GATA1 protein decreased by 30-43% (p=0.03) within 15 minutes and 66% by 90 minutes (similar decreases observed in cell lines), suggesting that heme disrupts GATA1 protein function resulting in the loss of autoregulation and reduced GATA1 mRNA. Of 88 genes in the ribosome pathway, 73 were significantly upregulated in Flvcr1-deleted cells, including 16 of the 17 ribosomal protein genes linked to Diamond-Blackfan anemia (DBA) or del(5q) myelodysplastic syndrome (MDS). When heme synthesis was induced in primary human erythroid marrow cells with ALA and iron, the transcription of ribosome protein genes such as Rps19, Rps14, and Rpl35 increased, further supporting the concept that heme assures sufficient ribosome production for globin protein synthesis. While P53 activation is a key factor in ineffective erythropoiesis caused by ribosomal protein imbalance (i.e., DBA and del(5q) MDS), GSEA did not reveal any increased activation of the P53 pathway in Flvcr1-deleted cells. To confirm that P53 was not involved in the ineffective erythropoiesis caused by excess heme, we generated mice lacking both P53 and FLVCR1. These double mutant mice had severe macrocytic anemia (HGB 2.4±0.70 g/dL; MCV 56.5±4.3 fL) comparable to mice lacking just FLVCR1. Thus, GATA1 turns on heme synthesis and initiates the erythroid differentiation program. GATA1 with heme assure each cell’s appropriate progression. Then heme turns off GATA1 to end differentiation. By linking excess heme to prematurely low GATA1, our data may also explain the ineffective (early termination of) erythropoiesis in DBA and reconcile the observations of Sci Transl Med 8:338ra67, 2016 and Nat Med 20:748, 2014. DisclosuresNo relevant conflicts of interest to declare.

Clonidine reduces norepinephrine and improves bone marrow function in a rodent model of lung contusion, hemorrhagic shock, and chronic stress

Propranolol has been shown previously to restore bone marrow function and improve anemia after lung contusion/hemorrhagic shock. We hypothesized that daily clonidine administration would inhibit central sympathetic outflow and restore bone marrow function in our rodent model of lung contusion/hemorrhagic shock with chronic stress. Male Sprague-Dawley rats underwent 6days of restraint stress after lung contusion/hemorrhagic shock during which the animals received clonidine (75μg/kg) after the restraint stress. On postinjury day 7, we assessed urine norepinephrine, blood hemoglobin, plasma granulocyte colony stimulating factor, and peripheral blood mobilization of hematopoietic progenitor cells, as well as bone marrow cellularity and erythroid progenitor cell growth. The addition of clonidine to lung contusion/hemorrhagic shock with chronic restraint stress significantly decreased urine norepinephrine levels, improved bone marrow cellularity, restored erythroid progenitor colony growth, and improved hemoglobin (14.1±0.6 vs 10.8±0.6g/dL). The addition of clonidine to lung contusion/hemorrhagic shock with chronic restraint stress significantly decreased hematopoietic progenitor cells mobilization and restored granulocyte colony stimulating factor levels. After lung contusion/hemorrhagic shock with chronic restraint stress, daily administration of clonidine restored bone marrow function and improved anemia. Alleviating chronic stress and decreasing norepinephrine is a key therapeutic target to improve bone marrow function after severe injury.

Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells

Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ(9) desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ(9)-desaturase inhibited cells 48 h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer.

Analysis and observation of T lymphocyte subsets and curative effects in patients with acquired pure red cell aplasia

Objectives To investigate distribution of T lymphocyte subsets, absolute value and proportion of large granular lymphocyte(LGL) in peripheral blood and bone marrow of acquired pure red cell aplasia (PRCA) patients, and to evaluate patients′ immune function and efficacy of immunosuppressive agents. Methods From January 2002 to November 2015, a total of 25 patients with acquired PRCA who were treated at Department of Hematology, People′s Hospital of Xinjiang Uygur Autonomous Region at first-visit were enrolled into this study, as study group. Inclusion criteria of study group: patients′ diagnoses were conformed to the diagnostic criteria of PRCA in Diagnosis and Curative Effect of Hematology (3rd edition) and clinical data were complete. Exclusion criteria: other anemia disease and clinical data were not complete. In 25 cases of PRCA patients, 5 cases were secondary PRCA with T cell-large granular lymphocyte leukemia (T-LGLL), other 20 cases were primary PRCA. And 25 cases of health people who were receiving physical examination at the People′s Hospital of Xinjiang Uygur Autonomous Region during the same period were recruited into control group randomly, by simple random method. Inclusion criteria of control group: no abnormality in blood routine examination and biochemical test. Exclusion criteria: abnormality in blood routine examination, and suffering from immune disease, tumor. T lymphocyte subsets of subjects in study group and control group were detected by flow cytometry. Blood routine examination and bone marrow examination were carried out in study group before and after treatment. Then absolute value and proportion of LGL in bone marrow, peripheral blood and curative effect after drug treatment of cyclosporine A and so on of 25 acquired PRCA patients were analyzed retrospectively and compared statistically. The study protocol was approved by the Ethical Review Board of Investigation in Human at People′s Hospital of Xinjiang Uygur Autonomous Region. There were no statistically significant differences of subjects′ clinical data, such as age and gender ratio, between two groups (P>0.05). Results ①Compared with control group, helper T lymphocyte (Th) proportion and Th/suppressor T lymphocyte (Ts) ratio of acquired PRCA patients in study group were significantly lower, Th proportion: (36.6±3.8)% vs (45.1±2.1)%; Th/Ts ratio: 1.2±0.2 vs 2.2±0.4; there were significant differences (t=9.161, 12.174, P=0.032, 0.021), and Ts proportion of study group was significantly higher than that of control group, (30.5±2.8)% vs (20.2±1.9)%; there was significant difference (t=13.460, P=0.021). There was no statistically significant difference of natural killer (NK) cell proportion between two groups, (11.3±1.8)% vs (10.3±1.3)%; and t=1.572, P=0.344. In study group, Th proportion, Th/Ts ratio of secondary PRCA patients with T-LGLL were lower than those of primary PRCA patients, Th proportion: (36.1±3.7)% vs (39.1±6.1)%; Th/Ts ratio: 1.0±0.2 vs 1.2±0.1; but the differences were not significant (t=2.293, 2.513; P=0.301, 0.297), and Ts proportion of secondary PRCA patients with T-LGLL was higher than that of primary PRCA patients, (31.0±2.7)% vs (28.4±2.0)%; but the difference was not significant (t=1.472, P=0.384). There was no statistically significant difference of NK cells proportion between two groups, (11.3±1.8)% vs (11.1±2.7)%; and t=1.572, P=0.364. ② Among 25 acquired PRCA patients in study group, 16 cases had higher proportion of lymphocytes in peripheral blood (64%, 16/25), and 10 cases had 2~3 granules LGL (40%, 10/25); 8 cases had more than 20% LGL of total lymphocytes (32%, 8/25). Four cases had 2~3 granules LGL in bone marrow (16%, 4/25); 3 cases had more than 20% LGL of total lymphocytes (12%, 3/25). The ratio of patients with LGL in peripheral blood was significantly higher than that in the bone marrow (χ2=6.595, P=0.003). ③ After treatment, hemogram of 18 cases returned to normal, including four cases with normal myelogram, fourteen cases with decreased bone marrow erythroid cell hyperplasia, which was higher than that before treatment. Effectiveness evaluation of study group was as follows: four PRCA patients were cured basically; fourteen cases achieved remission; six cases achieved significant progress; one case was invalid. The total effective rate of treatment was 96% (24/25). Five secondary PRCA patients with T-LGLL didn′t need blood transfusion after treatment, but unlike primary PRCA patients, their hemoglobin levels didn′t recover to normal reference range. Conclusions The disorder of T lymphocyte subsets in patients with acquired PRCA caused the disorder of cellular immune function, and LGL detected in peripheral blood would help the diagnosis of acquired PRCA. Effect of cyclosporin A treatment on acquired PRCA was very significant, but curative effect of secondary PRCA patients with T-LGLL was poorer than that of primary PRCA. Key words: Red-cell aplasia, pure; Anemia, aplastic; T-lymphocyte subsets; Leukemia, large granular lymphocytic; Cyclosporine

Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells

Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ9 desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ9-desaturase inhibited cells 48h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer

Bone marrow infiltration by multiple myeloma causes anemia by reversible disruption of erythropoiesis.

Multiple myeloma (MM) infiltrates bone marrow and causes anemia by disrupting erythropoiesis, but the effects of marrow infiltration on anemia are difficult to quantify. Marrow biopsies of newly diagnosed MM patients were analyzed before and after four 28-day cycles of non-erythrotoxic remission induction chemotherapy. Complete blood cell counts and serum paraprotein concentrations were measured at diagnosis and before each chemotherapy cycle. At diagnosis, marrow area infiltrated by myeloma correlated negatively with hemoglobin, erythrocytes, and marrow erythroid cells. After successful chemotherapy, patients with less than 30% myeloma infiltration at diagnosis had no change in these parameters, whereas patients with more than 30% myeloma infiltration at diagnosis increased all three parameters. Clinical data were used to develop mathematical models of the effects of myeloma infiltration on the marrow niches of terminal erythropoiesis, the erythroblastic islands (EBIs). A hybrid discrete-continuous model of erythropoiesis based on EBI structure/function was extended to sections of marrow containing multiple EBIs. In the model, myeloma cells can kill erythroid cells by physically destroying EBIs and by producing proapoptotic cytokines. Following chemotherapy, changes in serum paraproteins as measures of myeloma cells and changes in erythrocyte numbers as measures of marrow erythroid cells allowed modeling of myeloma cell death and erythroid cell recovery, respectively. Simulations of marrow infiltration by myeloma and treatment with non-erythrotoxic chemotherapy demonstrate that myeloma-mediated destruction and subsequent reestablishment of EBIs and expansion of erythroid cell populations in EBIs following chemotherapy provide explanations for anemia development and its therapy-mediated recovery in MM patients.

Cell cycle, proliferation and apoptosis in erythroblasts cultured from patients with β-thalassaemia major.

Cell cycle, proliferation and apoptosis in erythroblasts cultured from patients with β-thalassaemia major.

Evidence that the expression of transferrin receptor 1 on erythroid marrow cells mediates hepcidin suppression in the liver

Hepcidin is the key regulator of iron absorption and recycling, and its expression is suppressed by red blood cell production. When erythropoiesis is expanded, hepcidin expression decreases. To gain insight into the stage of erythroid differentiation at which the regulation might originate, we measured serum hepcidin levels in archived pure red cell aplasia samples from patients whose block in erythroid differentiation was well defined by hematopoietic colony assays and marrow morphologic review. Hepcidin values are high or high normal in pure red cell aplasia patients in whom erythropoiesis is inhibited prior to the proerythroblast stage, but are suppressed in patients with excess proerythroblasts and few later erythroid cells. These data suggest that the suppressive effect of erythropoietic activity on hepcidin expression can arise from proerythroblasts, the stage at which transferrin receptor 1 expression peaks, prompting the hypothesis that transferrin receptor 1 expression on erythroid precursors is a proximal mediator of the erythroid regulator of hepcidin expression. Our characterization of erythropoiesis, iron status, and hepcidin expression in mice with global or hematopoietic cell-specific haploinsufficiency of transferrin receptor 1 provides initial supporting data for this model. The regulation appears independent of erythroferrone and growth differentiation factor 15, supporting the concept that several mechanisms signal iron need in response to an expanded erythron.