Suppression Of Erythropoiesis Research Articles

Supplementation of Chlorella vulgaris Ameliorates the Stress-induced Hematological Alterations in Wistar Rats

Stress has been associated with various diseases and physiological disruptions. Chlorella vulgaris is known for its antioxidant properties. This study examined the effects of C. vulgaris on the hematological profile of stress-induced Wistar rats. Stress significantly impacted various hematological parameters, including erythrocyte count, hemoglobin concentration, hematocrit levels, white blood cell count, and platelet count. The supplementation of cultivated C. vulgaris effectively restored erythrocyte count and suppressed elevated lymphocyte levels, while commercially available C. vulgaris and amitriptyline drugs had no significant effects on these parameters. Stress caused smaller-sized erythrocytes but did not significantly affect mean corpuscular volume. Both amitriptyline and C. vulgaris restored platelet levels, while mean platelet volume remained unaffected. Overall, C. vulgaris showed promise as a therapeutic intervention for countering stress-induced inhibition of erythropoiesis and restoring erythrocyte count, but more research is needed to understand the underlying mechanisms and develop effective strategies for managing stress-related changes in hematological parameters.

Hemolysis-driven IFNα production impairs erythropoiesis by negatively regulating EPO signaling in sickle cell disease

AbstractDisordered erythropoiesis is a feature of many hematologic diseases, including sickle cell disease (SCD). However, very little is known about erythropoiesis in SCD. Here, we show that although bone marrow (BM) erythroid progenitors and erythroblasts in Hbbth3/+ thalassemia mice were increased more than twofold, they were expanded by only ∼40% in Townes sickle mice (SS). We further show that the colony-forming ability of SS erythroid progenitors was decreased and erythropoietin (EPO)/EPO receptor (EPOR) signaling was impaired in SS erythroid cells. Furthermore, SS mice exhibited reduced responses to EPO. Injection of mice with red cell lysates or hemin, mimicking hemolysis in SCD, led to suppression of erythropoiesis and reduced EPO/EPOR signaling, indicating hemolysis, a hallmark of SCD, and could contribute to the impaired erythropoiesis in SCD. In vitro hemin treatment did not affect Stat5 phosphorylation, suggesting that hemin-induced erythropoiesis suppression in vivo is via an indirect mechanism. Treatment with interferon α (IFNα), which is upregulated by hemolysis and elevated in SCD, led to suppression of mouse BM erythropoiesis in vivo and human erythropoiesis in vitro, along with inhibition of Stat5 phosphorylation. Notably, in sickle erythroid cells, IFN-1 signaling was activated and the expression of cytokine inducible SH2–containing protein (CISH), a negative regulator of EPO/EPOR signaling, was increased. CISH deletion in human erythroblasts partially rescued IFNα-mediated impairment of cell growth and EPOR signaling. Knocking out Ifnar1 in SS mice rescued the defective BM erythropoiesis and improved EPO/EPOR signaling. Our findings identify an unexpected role of hemolysis on the impaired erythropoiesis in SCD through inhibition of EPO/EPOR signaling via a heme-IFNα-CISH axis.

Intracerebral Hemorrhage Leads to Anemia By Disrupting Erythropoiesis and Erythromyeloblastic Islands

Intracerebral hemorrhage (ICH), caused by the rupture of a cerebral vessel and subsequent entry of blood into the brain parenchyma, is a major form of acquired brain injury and contributes a remarkable proportion of stroke-related morbidity and mortality. Due to the few proven effective emergency and preventive treatments, patients who survived suffer varying degrees of poor prognosis that affect their quality of life. There is an urgent need to explore the cellular and molecular basis of the differences in the severity and prognosis of ICH. Anemia is a common manifestation of ICH. However, the mechanisms underlying ICH-induced anemia as we as the correlation between anemia and ICH progression/prognosis remain unclear. In this study, we found that approximately 35.1% (39 of 111) of ICH patients exhibited anemia and that decreased hemoglobin levels were positively associated with higher scores on the National Institutes of Health Stroke Scale, which quantifies the degree of neurological deficit, at admission. Furthermore, patients with decreased hemoglobin levels at admission showed higher modified Rankin scale scores at 3 months, which reflect the global disability of daily living at this time, suggesting that anemia was associated with a more severe progression and poor patient prognosis. To investigate the effects of ICH on bone marrow (BM) erythropoiesis, we conducted experiments in 12-month-old mice with collagenase-induced striatal ICH. Our results showed that ICH caused apparent anemia on day 3, associated with larger hematomas in the striatum and more severe neurologic deficits. We first evaluated the phenotype of hematopoietic progenitors and found that mice suffering ICH exhibited increased granulocyte monocyte progenitors but a significant decrease in common myeloid progenitors and megakaryocyte erythrocyte progenitors compared to control mice, suggesting a reduced source of erythroid progenitors. Further analysis revealed that the colony-forming ability of erythroid progenitors and subsequent terminal differentiation was significantly suppressed after ICH. To assess whether ICH-induced suppression of erythropoiesis is associated with erythromyeloblastic islands (EMBIs), the niche for erythropoiesis/myelopoiesis, we performed ICH using 12-month-old EpoR-tdTomato-Cre mice and found that the number of BM EMBIs and EpoR + macrophages decreased significantly. Moreover, the number and proportion of developing erythroid cells surrounding EMBIs decreased while LY6G + cells representing granulocytes increased prominently, indicating an altered microenvironment, with EMBI favoring granulopoiesis over erythropoiesis after ICH. Studies have shown that inflammation plays a crucial role in secondary brain injury after ICH. We hypothesize that inflammatory cytokines may contribute to the suppression of erythropoiesis observed in ICH mouse models. To test this, we measured inflammatory factors in both peripheral blood and BM fluid using Quantibody Mouse Inflammation Array. In particular, IL-6 concentrations in peripheral blood and BM fluid in ICH mice were significantly higher than controls. Treatment with MR16-1, an IL-6 antagonist, alleviated ICH-induced anemia and improved BM erythropoiesis in ICH mice, as evidenced by increased peripheral red blood cells and hemoglobin levels, elevated numbers of erythroid progenitors and terminal erythroblast. MR16-1 treatment also improved BM EMBI formation and increased EpoR + macrophages. In addition, MR16-1 treatment effectively improved neurologic deficits and reduced brain hematoma after ICH. Administration of recombinant IL-6 to wild-type mice resulted in decreased levels of red blood cells and hemoglobin along with suppression of erythropoiesis in the BM. Collectively, these results suggest that ICH modulates erythropoiesis and erythromyeloblastic islands function to ultimately lead to anemia and reveal a possible therapeutic opportunity to reverse ICH-induced anemia and improve ICH prognosis by targeting IL-6.

Is Epigenetic Modulation to Induce Fetal Hemoglobin Translatable in Sickle Cell Mice?

Sickle cell disease (SCD) is a genetic disorder caused by a mutation in the β-globin subunit of hemoglobin, resulting in the formation of HbS. Under deoxygenation, HbS polymerizes leading to red blood cell sickling, increased hemolysis and multi-organ damage. During human fetal development, due to expression of the γ-globin gene, the predominant type of hemoglobin produced is fetal hemoglobin (HbF). However, after birth, this shifts to adult globin (HbA or HbS in SCD). This is driven by epigenetic repression of γ-globin due to an increased methylation of its gene promoters. DNA methyltransferase 1 (DNMT1) is an important enzyme for DNA methylation and is known to be pivotal in regulation of HbF expression. Reactivation of HbF provides a functional hemoglobin, and therefore has the potential to treat SCD symptoms ( Steinberg et al. JAMA 2003;289:1645-51). HbF induction is an accepted treatment strategy in SCD; even small increases result in decreased vaso-occlusive crises and mortality ( Kato et al. Nat Rev Dis Primers 2018;4:18010). Decitabine inhibits DNMT1 activity, thus promoting γ-globin expression and thereby increasing HbF levels. While inhibition of DNA methylation is known to cause HbF induction in humans and non-human primates, the effects observed in SCD mouse models are more ambiguous. This study investigated to which extent decitabine treatment could induce HbF expression in the Townes SCD model. In Townes mice, the murine globin genes have been substituted for their sickle human counterparts, HBA, HBB sickle and HBG1, and mimic many features of human SCD. Mice aged 4-5 weeks were dosed subcutaneously 3 times a week for 12 weeks with research-grade decitabine (0.6 mg/kg or 0.4 mg/kg) or vehicle. Blood samples were collected for analysis of HbF protein levels by HPLC (represented as % of total Hb) and F-cells by flow cytometry (represented as % of the total RBC population positive for HbF), complete blood counts, histology, and other markers of disease and organ damage. Results are presented as mean ± SD. Groups were compared using an ANCOVA model, adjusting for multiple comparisons (Dunnett) with p<0.05 considered statistically significant. For both treatment groups, HbF was significantly elevated compared to the control group, with the maximal response observed at 12 weeks (Figure 1). F-cells at the end of the study were increased in both 0.4 mg/kg (11.4±1.5%) and 0.6 mg/kg (11.8±1.4%) groups compared to vehicle (4.5±0.6%). The same was observed for protein levels: 0.4 mg/kg group (1.2±0.3%), 0.6 mg/kg (1.4±0.3%) compared to vehicle (0.4±0.1%). Interestingly, after 12 weeks RBC and reticulocyte counts were significantly decreased in treatment groups and mean corpuscular volume (MCV) was significantly increased. No changes on LDH, bilirubin and liver markers (ALP, ALT, AST) were observed at 6 or 12 weeks. The results showed that treatment groups had significantly higher HbF levels than the vehicle group. However, the level of induction was lower than observed for inhibition of DNA methylation in humans and primates. The decrease in RBCs and reticulocytes could be partially explained by a combination of HbF induction, reduction of sickle RBC, and inhibition of erythropoiesis. The lack of changes in disease markers suggests that the increase in HbF levels in Townes mice might not be sufficient to alleviate the disease. This observation is consistent with recently published data (Woodard et al. Dis Model Mech. 2022;15(6)) that indicates that Townes mice are less suitable for studying this mechanism of action since, even though they have all human globin genes and some proximal regulatory elements, they lack key cis-acting distant DNA elements that regulate the globin genes. However, in primates the globin structure and regulation mechanisms are similar to human (Hardison. Cold Spring Harb Perspect Med. 2012;2(12)). In conclusion, although the measured levels of HbF protein were no higher than 2%, we found that decitabine can induce HbF expression in the Townes mouse model of SCD. However, within the 12-weeks, there was no clear beneficial effect on the disease markers studied. The successful induction of HbF suggests that better outcomes might be achieved with further optimization of the dosing and duration of the study. This indicates that SCD mice may be useful for preclinical studies of HbF induction, but caution is needed when extrapolating results to humans.

Phase 1b/2 Study Evaluating the Safety and Efficacy of Canakinumab with Darbepoetin Alfa in Patients with Lower-Risk Myelodysplastic Syndromes (MDS) Who Have Failed Erythropoietin Stimulating Agents (ESA)

Introduction: Pyroptosis is a form of inflammatory, caspase-1 dependent lytic cell death mediated by inflammasome activation and ultimately leading to enumeration of pro-inflammatory cytokines, particularly interleukin 1β (IL-1β) and is a key driver of MDS pathogenesis (Basiorka A, Blood 2016). Release of IL-1β by pyroptotic cells leads to paracrine stimulation of neighboring hematopoietic and stromal cells resulting in amplification of this circuit, myeloid derived suppressor cell (MDSC) accumulation as well as direct suppression of erythropoiesis. Thus, we investigated the combination of the IL-1β neutralizing antibody canakinumab in combination with darbepoetin in patients with ESA refractory lower risk MDS and present the final dose phase 1b data. Methods: Patients ≥ 18 years old with IPSS-R very low to intermediate risk MDS who were ESA refractory and hypomethylating agent (HMA) naïve with adequate organ function were eligible. Patients must have been RBC transfusion dependent or hemoglobin < 9 gm/dl with symptoms. Treatment consisted of subcutaneous (SC) canakinumab on day 1 of a 28 cycle (150mg dose level 1, 300mg dose level 2) and darbepoetin SC 300µg days 1 and 15 of each cycle for a total of 6 cycles with continuation of therapy for responding patients. The phase 1b portion utilized a 3+3 dose escalation design with 6 patients treated at the maximum tolerated dose (MTD) to define the recommended phase 2 dose (RP2D). Bone marrow (BM) disease assessments occurred every 3 cycles with response defined by IWG 2018 criteria. Quality of life was assessed by QUALMS (Abel A, Haematologica, 2016). Peripheral blood mononuclear cells (PBMCs) were obtained at disease assessments and day 15 of C1 for evaluation of adapter protein apoptosis associated speck-like protein containing a CARD (ASC) specks as a biomarker of inflammasome formation with quantification as previously described (McLemore A, Pyroptosis: Methods and Protocols, 2023). Total IL-1β of BM plasma was assayed by ELISA. Results: Nine patients enrolled with a median age of 71 (range: 61-85 years), 67% female with baseline IPSS-R, IPSS-M and mutation data shown in Table. By IPSS-M, 3, 3, 1, 2 patients were low, moderately-low, moderately high, and high-risk categories. All IPSS-M lower risk mutant patients had splicing mutations. The median number of mutations was 2 (range 0-5). 100% of patients were ESA refractory. Additionally, 33% and 56% were refractory to lenalidomide and luspatercept therapy, respectively. Eight (89%) patients were PRBC dependent at baseline (56% [n=5] with high transfusion burden). The median follow-up duration was 6.1 months (range 3-17.8). The most common adverse events were neutropenia (n=7), followed by thrombocytopenia (n=4), and abnormal liver function tests (n=4). There were two grade 3 adverse events including thrombocytopenia and colitis, both unrelated to study treatment. There were no dose-limiting toxicities or serious adverse events in the phase 1 portion. All patients achieved the best response as stable disease, and no one had progression disease. No patients achieved objective response or significant reduction in transfusion burden. There was no statistical improvement in total QUALMS score from baseline to end of treatment (P=0.72) nor in the individual components (i.e. physical burden, benefit finding and emotional burden, P=0.69, P=0.91, P=0.630; respectively) although several patients had individual improvements. The percentage of PBMCs with ASC specks at baseline ranged from 0.3945-2.978% (median 1.93%). ASC specks decreased significantly after cycle 1 of therapy and were durably suppressed over the course of treatment in a majority of patients (Figure). Extracellular IL-1β in bone marrow plasma robustly increased with treatment and remained elevated (representing non-functional IL-1β [canakinumab bound IL-1β]; P=0.002 cycle 6 versus baseline), supporting pharmacodynamic effect with no differences observed between DL1 and DL2. Conclusions: The combination of canakinumab and darbepoetin was safe and well tolerated and 300mg of canakinumab is the RP2D. Canakinumab inhibited inflammasome activation as evidenced by a reduction of ASC specks although this did not translate to clinical responses. Phase 2 of the study will focus on patients with lower transfusion burden and molecular subsets with highest IL-1β expression (e.g. TET2/ DNMT3A mutant).

Increased blood reactive oxygen species and hepcidin in obstructive sleep apnea precludes expected erythrocytosis.

Obstructive sleep apnea (OSA) causes intermittent hypoxia during sleep. Hypoxia predictably initiates an increase in the blood hemoglobin concentration (Hb); yet in our analysis of 527 patients with OSA, >98% did not have an elevated Hb. To understand why patients with OSA do not develop secondary erythrocytosis due to intermittent hypoxia, we first hypothesized that erythrocytosis occurs in these patients, but is masked by a concomitant increase in plasma volume. However, we excluded that explanation by finding that the red cell mass was normal (measured by radionuclide labeling of erythrocytes and carbon monoxide inhalation). We next studied 45 patients with OSA before and after applying continuous positive airway pressure (CPAP). We found accelerated erythropoiesis in these patients (increased erythropoietin and reticulocytosis), but it was offset by neocytolysis (lysis of erythrocytes newly generated in hypoxia upon return to normoxia). Parameters of neocytolysis included increased reactive oxygen species from expanded reticulocytes' mitochondria. The antioxidant catalase was also downregulated in these cells from hypoxia-stimulated microRNA-21. In addition, inflammation-induced hepcidin limited iron availability for erythropoiesis. After CPAP, some of these intermediaries diminished but Hb did not change. We conclude that in OSA, the absence of significant increase in red cell mass is integral to the pathogenesis, and results from hemolysis via neocytolysis combined with inflammation-mediated suppression of erythropoiesis.

Contributions of bone marrow monocytes/macrophages in myeloproliferative neoplasms with JAK2V617F mutation.

The classic BCR-ABL1-negative myeloproliferative neoplasm (MPN) is a highly heterogeneous hematologic tumor that includes three subtypes, namely polycythemia vera (PV), essential thrombocytosis (ET), and primary myelofibrosis (PMF). Despite having the same JAK2V617F mutation, the clinical manifestations of these three subtypes of MPN differ significantly, which suggests that the bone marrow (BM) immune microenvironment may also play an important role. In recent years, several studies have shown that peripheral blood monocytes play an important role in promoting MPN. However, to date, the role of BM monocytes/macrophages in MPN and their transcriptomic alterations remain incompletely understood. The purpose of this study was to clarify the role of BM monocytes/macrophages in MPN patients with the JAK2V617F mutation. MPN patients with the JAK2V617F mutation were enrolled in this study. We investigated the roles of monocytes/macrophages in the BM of MPN patients, using flow cytometry, monocyte/macrophage enrichment sorting, cytospins and Giemsa-Wright staining, and RNA-seq. Pearson correlation coefficient analysis was also used to detect the correlation between BM monocytes/macrophages and the MPN phenotype. In the present study, the proportion of CD163+ monocytes/macrophages increased significantly in all three subtypes of MPN. Interestingly, the percentages of CD163+ monocytes/macrophages are positively correlated with HGB in PV patients and PLT in ET patients. In contrast, the percentages of CD163+ monocytes/macrophages are negatively correlated with HGB and PLT in PMF patients. It was also found that CD14+CD16+ monocytes/macrophages increased and correlated with MPN clinical phenotypes. RNA-seq analyses demonstrated that the transcriptional expressions of monocytes/macrophages in MPN patients are relatively distinct. Gene expression profiles of BM monocytes/macrophages suggest a specialized function in support of megakaryopoiesis in ET patients. In contrast, BM monocytes/macrophages yielded a heterogeneous status in the support or inhibition of erythropoiesis. Significantly, BM monocytes/macrophages shaped an inflammatory microenvironment, which, in turn, promotes myelofibrosis. Thus, we characterized the roles of increased monocytes/macrophages in the occurrence and progression of MPNs. Our findings of the comprehensive transcriptomic characterization of BM monocytes/macrophages provide important resources to serve as a basis for future studies and future targets for the treatment of MPN patients.

Prevalence of anemia and its predictors among patients with chronic kidney disease admitted to a teaching hospital in Ethiopia: A hospital-based cross-sectional study.

Anemia is a common complication of chronic kidney disease (CKD) and is associated with adverse patient outcomes. However, data on the prevalence of anemia in CKD patients is sparse, particularly in resource-limited settings. Therefore, this study aimed to assess the prevalence of anemia and its predictors among patients with CKD admitted to the Jimma medical center, southwest Ethiopia. A hospital-based prospective cross-sectional study was conducted from September 1 to November 30, 2020. All adult patients with CKD aged ≥18 years who fulfilled the inclusion criteria were consecutively recruited into the study. Data were entered into the Epi data manager version 4.4.1 and then exported to SPSS version 22 (IBM Corp., Armonk, NY) for analysis. The predictors of anemia were determined using multivariable logistic regression analysis. Statistical significance was set at P < .05. A total of 150 patients were included in this study. Of these, 64.67% were male, 56.67% had stage 5 CKD, 78% had a CKD duration of less than 1 year, and 74% had proteinuria. Hypertension (40.7%) and diabetes (14.7%) were the common causes of CKD. The prevalence of anemia was 85.33%. Of the patients, 28.67%, 40.67%, and 16% had mild, moderate, and severe anemia, respectively. On multivariate logistic regression, stage 4 CKD (adjusted odds ratio [AOR] 3.2, confidence interval [CI]: 1.78-12.91, P = .025), stage 5 CKD (AOR 4.03, CI: 1.17-13.73, P = .016), and CKD duration of less than 1 year (AOR 3, CI: 1.19-9.11, P = .007) were significantly associated with anemia. The prevalence of anemia among stage 3 to 5 CKD patients was very high. Anemia was significantly associated with the severity and duration of CKD. Therefore, serial follow-up of patients with a long duration and advanced stages of CKD may help prevent anemia and its adverse consequences.

The Effect of Monoferric Transferrins on Hypoferremia in a Murine Model of Inflammation

Introduction: Modulation of iron metabolism and suppression of erythropoiesis are central components of the acute phase response (APR) and underlie the anemia of inflammation. Hypoferremia is characteristic of the APR and contributes to erythroid suppression by restricting the iron available for erythropoiesis. The role of transferrin (Tf) signaling in the APR has not been explored. We previously generated mice with transferrin mutations that prevent binding of iron to either the N-terminal (N-blocked) or C-terminal (C-blocked) lobe and demonstrated distinct effects on erythropoiesis and the status of the iron regulatory hormone hepcidin. We hypothesize that monoferric transferrins differentially affect modulation of iron metabolism during the APR. Methods: For a model of acute inflammation, 14 day old wild-type (wt), N-blocked and C-blocked Tf mutant mice were treated with 1mg/kg lipopolysaccharide (LPS) or carrier by intraperitoneal injection and sacrificed 6 hours later. A repeat dose regimen, consisting of seven consecutive daily intraperitoneal injections of 0.3 mg/kg LPS or carrier beginning at seven days of age, was used as a model of chronic inflammation. Results: In the acute model, wt and C-blocked mice demonstrate the expected decrease in serum iron in response to LPS administration (38 + 7 and 70 +6 ug/dL, respectively). In contrast, N-blocked mice fail to decrease serum iron following LPS administration (164 + 22 ug/dL; p <0.01). Liver and spleen iron concentrations are also significantly higher in N-blocked mice following LPS administration, compared to C-blocked and wild-type mice (LIC: 587 + 28 vs 311 + 53 and 243 + 9 ug Fe/g dry weight; p <0.0001; SIC: 471 + 16 vs 406 + 16 and 336 + 18 ug Fe/g dry weight; p <0.05). LPS administration increases hepcidin all three strains. In contrast, elevated Hamp1 expression is not sustained in the repeat dose model. Nevertheless, serum iron remains elevated in N-blocked mice (201.3 + 30 ug/dL; p <0.05) whereas it returns to baseline in wt and C-blocked mice (76.8 + 39 and 86.6 + 21.4 ug/dL, respectively). Liver and spleen iron concentrations also remain higher in the N-blocked mice relative to C-blocked and wt mice (764.7 + 202 vs 295.7 + 62.7 and 301.3 + 74.8 ug Fe/g dry weight; p <0.05 for liver and 511.9 + 36.2 vs 404.8 + 61.3 and 427.3 + 33.ug Fe/g dry weight; p < 0.05 for spleen). All mice demonstrate marked splenomegaly in response to repeated doses of endotoxin without significant differences between strains. Hemoglobin concentrations are decreased in all strains after repeated endotoxin dosing, with N-blocked mice presenting the most severe anemia (4.1 + .21 vs 5.1 + .42 and 5.3 + .28 g/dL for N-blocked, C-blocked and wt, respectively; p < 0.05). Conclusions: In LPS-mediated inflammation, mice unable to bind iron on the N-lobe of transferrin fail to develop hypoferremia and have elevated liver iron concentrations, despite increased hepcidin and splenic iron concentrations. We speculate that the iron excess observed in the Tf N-blocked mice arises from a more profound suppression of erythropoiesis, and relatively decreased utilization of iron in hemoglobin production. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Erythropoiesis and Malaria, a Multifaceted Interplay.

One of the major pathophysiologies of malaria is the development of anemia. Although hemolysis and splenic clearance are well described as causes of malarial anemia, abnormal erythropoiesis has been observed in malaria patients and may contribute significantly to anemia. The interaction between inadequate erythropoiesis and Plasmodium parasite infection, which partly occurs in the bone marrow, has been poorly investigated to date. However, recent findings may provide new insights. This review outlines clinical and experimental studies describing different aspects of ineffective erythropoiesis and dyserythropoiesis observed in malaria patients and in animal or in vitro models. We also highlight the various human and parasite factors leading to erythropoiesis disorders and discuss the impact that Plasmodium parasites may have on the suppression of erythropoiesis.

Arsenic impairs the lineage commitment of hematopoietic progenitor cells through the attenuation of GATA-2 DNA binding activity

Arsenic exposure produces significant hematotoxicity in vitro and in vivo. Our previous work shows that arsenic (in the form of arsenite, AsIII) interacts with the zinc finger domains of GATA-1, inhibiting the function of this critical transcription factor, and resulting in the suppression of erythropoiesis. In addition to GATA-1, GATA-2 also plays a key role in the regulation of hematopoiesis. GATA-1 and GATA-2 have similar zinc finger domains (C4-type) that are structurally favorable for AsIII interactions. Taking this into consideration, we hypothesized that early stages of hematopoietic differentiation that are dependent on the function of GATA-2 may also be disrupted by AsIII exposure. We found that in vitro AsIII exposures disrupt the erythromegakaryocytic lineage commitment and differentiation of erythropoietin-stimulated primary mouse bone marrow hematopoietic progenitor cells (HPCs), producing an aberrant accumulation of cells in early stages of hematopoiesis and subsequent reduction of committed erythro-megakaryocyte progenitor cells. Arsenic significantly accumulated in the GATA-2 protein, causing the loss of zinc, and disruption of GATA-2 function, as measured by chromatin immunoprecipitation and the expression of GATA-2 responsive genes. Our results show that the attenuation of GATA-2 function is an important mechanism contributing to the aberrant lineage commitment and differentiation of early HPCs. Collectively, findings from the present study suggest that the AsIII-induced disruption of erythro-megakaryopoiesis may contribute to the onset and/or exacerbation of hematological disorders, such as anemia.

Red blood cell distribution width as a marker of hyperinflammation and mortality in COVID-19.

Red blood cell distribution width (RDW) could reflect interleukin-6 (IL-6) systemic activity since anisocytosis represents the inhibition of erythropoiesis, leaded by the hyperinflammatory background. Our objective was to analyze RDW performance to predict outcome in coronavirus disease 2019 (COVID-19) acute respiratory distress syndrome (ARDS). Retrospective observational study including 173 patients with COVID-19-associated ARDS. Data was analyzed at hospital admission, inclusion in the TOCICOV Study (day 0), days 1, 3, 7 and 15 post-inclusion. Overall, 57% patients received tocilizumab. Overall mortality was 20.8%. RDW was higher in non-survivors compared to survivors at admission (13.53% vs. 14.35, P=0.0016), day 0 (13.60% vs. 14.42, P=0.026), day 3 (13.43% vs. 14.36, P<0.001) and day 7 (13.41% vs. 14.31, P=0.046), presenting better discrimination ability for mortality than other prognostic markers [area under the curve-receiver operating characteristic (AUC-ROC) =0.668 for admission RDW, 0.680 for day 0 RDW, 0.695 for day 3 RDW and 0.666 for day 7 RDW]. RDW values did not vary significantly according to tocilizumab treatment. When adjusted by hemoglobin and tocilizumab treatment, only RDW at admission, day 0, day 3 and C reactive protein (CRP) at day 0 and day 1 were associated with mortality (P<0.05). Only in non-tocilizumab treated patients, IL-6 levels at day 0 were correlated with day 3 RDW (r=0.733, P=0.004) and with day 3 CRP (r=0.727, P=0.022). Both parameters showed significant statistical correlation (r=0.255 for day 1 RDW and CRP in the overall cohort and r=0.358 for day 3 RDW and CRP in patients not treated with tocilizumab, P<0.015). RDW predicts COVID-19-associated ARDS mortality and reflects the hyperinflammatory background and the effects of cytokines such as IL-6, irrespective of tocilizumab treatment.

Hydroxyurea Decouples Persistent F-Cell Elevation and Induction of γ-Globin

Mechanisms that control the fetal-to-adult hemoglobin switch are attractive therapeutic targets in sickle cell disease. In this study, we investigated developmental γ-globin silencing in the Townes humanized knock-in mouse model, which harbors a construct containing the human γ-, βA-, and βS-globin genes, and examined the utility of this model in evaluation of pharmacologic induction of fetal hemoglobin (HbF). We studied mouse pups on the day of delivery (P0) to 28 days after birth (P28). Regardless of the hemoglobin genotype (SS, AS, or AA), the proportion of F cells in peripheral blood was 100% at P0, declined sharply to 20% at P2, and was virtually undetectable at P14. Developmental γ-globin silencing in Townes mice was complete at P4 in association with significantly increased BCL11A expression in the primary erythropoietic organs of the mouse. Hydroxyurea given at P2 significantly sustained elevated percentages of F cells in mice at P14. However, the percentage of F cells declined at P14 for treatment begun at P4. A lack of augmentation of γ-globin mRNA in erythroid tissues suggests that the apparent increase in HbF in red cells caused by hydroxyurea was not due to sustained or re-activation of γ-globin transcription, but was instead a function of erythropoiesis suppression. Thus, we provide new details of the hemoglobin switch in the Townes murine model that recapitulates postnatal γ- to β-globin switch in humans and identify the myelosuppressive toxicity of hydroxyurea as a superseding factor in interpreting pharmacologic induction of HbF.

Effective Management of Non-Surgical Periodontal Treatment in a Patient with Severe Gingival Enlargement and Periodontal Bone Loss: A Case Report

The immune-inflammatory response of periodontal tissue can be influenced by various systemic variables such as hormone disorders, hematologic diseases, nutrition, and medications. Inflammatory cytokines released by infected periodontal tissue are implicated in suppressing erythrocyte growth, differentiation, and erythropoiesis inhibition. We present a case of non-surgical periodontal treatment in a 31-year-old female patient with severe gingival enlargement associated with aggressive bone destruction combined with iron deficiency anemia (IDA). The periodontal treatment plan was achieved in collaboration with the hematologist, starting with oral hygiene instruction, scaling and root planning (SRP) by sextants with systemic antibiotic therapy combined with a chemical agent and low-level-laser-therapy (LLLT), and extraction of hopeless teeth. Effective non-surgical periodontal treatment with SRP and LLLT reduces inflammation and gingival enlargements and controls the recolonization of subgingival sites by periodontal pathogens as a crucial factor to proceed with periodontal surgery. Function, aesthetics, and the patient’s social communication are all improved.

The association between Parvovirus B19 infection and persistent anaemia in renal transplant recipients

BackgroundThe immunosuppressive drug therapy makes post-transplant recipients are at risk of developing both primary viral diseases and reactivation of persistent viruses. There are published case reports and studies on Parvovirus B19 infection in organ transplant recipients which have led to persistent anaemia secondary to lengthy suppression of erythropoiesis. ObjectiveTo assess the association between persistent unexplained anaemia and Parvovirus B19 infection amongst renal transplant recipients MethodsA matched case-control study was conducted in five government transplant centres in Sri Lanka by recruiting 42 cases and 42 controls prospectively. Cases were defined as adult renal transplant recipients with moderate to severe persistent unexplained anaemia. Quantitative PCR assay was performed for the quantification of Parvovirus B19 DNA in plasma of cases and controls and positive samples were further tested for the presence of IgG antibodies against Parvovirus B19. ResultsPost-transplant unexplained persistent anaemia was identified in 5.6% (95% CI: 4.21–7.42%) of the screened population. Parvovirus B19 infection was detected in one patient amongst 42 cases (2.2%), and no controls had confirmed Parvovirus B19 infection. The odds ratio for the association between persistent anaemia (outcome) and Parvovirus B19 infection (exposure) was 3.0723 (95% CI: 0.1216–77.5982) for persistent anaemia and parvoviraemia. (P-value: 0.4957). ConclusionThe finding of 5.6% of post-transplant unexplained persistent anaemia amongst RTRs demonstrates its importance as a significant modifiable morbidity factor in the modern renal transplant era. The association between Parvovirus B19 infection and persistent anaemia in Sri Lankan renal transplant recipients was not demonstrated by this study.

Hematopoiesis, Inflammation and Aging-The Biological Background and Clinical Impact of Anemia and Increased C-Reactive Protein Levels on Elderly Individuals.

Anemia and systemic signs of inflammation are common in elderly individuals and are associated with decreased survival. The common biological context for these two states is then the hallmarks of aging, i.e., genomic instability, telomere shortening, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion and altered intercellular communication. Such aging-associated alterations of hematopoietic stem cells are probably caused by complex mechanisms and depend on both the aging of hematopoietic (stem) cells and on the supporting stromal cells. The function of inflammatory or immunocompetent cells is also altered by aging. The intracellular signaling initiated by soluble proinflammatory mediators (e.g., IL1, IL6 and TNFα) is altered during aging and contributes to the development of both the inhibition of erythropoiesis with anemia as well as to the development of the acute-phase reaction as a systemic sign of inflammation with increased CRP levels. Both anemia and increased CRP levels are associated with decreased overall survival and increased cardiovascular mortality. The handling of elderly patients with inflammation and/or anemia should in our opinion be individualized; all of them should have a limited evaluation with regard to the cause of the abnormalities, but the extent of additional and especially invasive diagnostic evaluation should be based on an overall clinical evaluation and the possible therapeutic consequences.

A rapid spectrophotometric method to identify inhibitors of human erythropoiesis

Erythropoiesis is a complex physiological process by which erythroid progenitors proliferate and differentiate into nonnucleated red blood cells. Several methods can be used to monitor in vitro the differentiation of erythroid precursors, and hence the toxic effects of drugs, chemicals, or pollutants. One of the most commonly available assay of erythropoiesis is the microscopic observation of differentiated cells after benzidine staining, which forms a blue complex with hemoglobin. However, this method is laborious and does not provide accurate results since it heavily relies on the reader's interpretation. Moreover, benzidine is a carcinogen and a highly reactive molecule which forces the reader to microscopically count differentiated and non-differentiated cells within a short time frame (5 min).Here we have developed a simple, inexpensive, in-vitro spectrophotometric assay to measure erythroid differentiation using K562 cell line as a model. Materials needed included 96-well round-bottomed microplates and a microplate reader. Remarkably, carcinogenic benzidine was replaced by its isomeric tetramethyl derivative, the 3,3′, 5,5′- tetramethylbenzidine (TMB), which presents several advantages: it is cheap, not mutagenic and a ready-to-use chromogenic substrate. A small volume (50 μl) of TMB added to the samples forms a blue complex in 15 min, and the reaction can be easily stopped and stabilized by the addition of H2SO4. The yellow precipitate is then solubilized, and the absorbance is measured at 450 nm. In addition, the suitability of the assay to determine the effects of compounds on erythroid differentiation was further tested with known inhibitors (artemisinin derivatives) of K562 differentiation.Overall, the reported methodology permits to measure in an accurate and reproducible manner the K562 differentiation and can be used for medium throughput screenings (MTS) of compounds or environmental toxics with potential erythro-toxicity and ability to inhibit erythroid differentiation.

Title: Efficacy and Safety of Ferric Derisomaltose for Treatment of Anemia in Chronic Kidney Disease Patients: A Systematic Review

BackgroundAnemia in chronic kidney disease (CKD) patients develop primarily due to iron and erythropoietin deficiency, factors contributing to this include uremia induced inhibition of erythropoiesis, shortened red cell survival and nutritional deficiency. Erythropoietin stimulating agents (ESA) used in CKD increases iron demand for the production of new red blood cells (RBCs). Oral iron does not provide a reliable route to meet the demand of the iron required and poses the risk of side effects. The intravenous formulation provides the fastest route, although multiple intravenous formulations are available they require multiple dosing and still carry a risk of an adverse event. Ferric derisomaltose/iron isomaltoside (IIM) is an intravenous formulation of iron recently approved by the Food and drug administration (FDA) for patients intolerant to oral iron and non-dialysis dependent CKD patients. FDI is administered in high dose usually in a single administration. In this systematic review, we evaluated the published literature for the efficacy and safety of ferric derisomaltose in both dialysis-dependent and dialysis independent patients.Material/MethodsA literature search was done using the following databases: PubMed, Cochrane, Embase, Clinical trials.gov, and Web of Science. The search was completed without using any filter and we used the Mesh Terms for “anemia”, “iron deficiency anemia” “chronic kidney disease” and “ferric compounds”. Total of 316 which were further screened and we included 2 trials, and 2 prospective studies. Case reports, preclinical trials, meta-analyses, and review articles were excluded. We followed the PRISMA guidelines for literature search and selection of studies. We only included the trial studies that were completed. We excluded the studies that did not report efficacy and safety.ResultsIn total, amongst the four studies, 1558 CKD patients received ferric derisomaltose in a single dose. Patients in all stages of CKD were studied but most patients were between stages I to stage III CKD, stage IV and V(dialysis-dependent) were studied by Kalra et al (2020). A single dose of 1000 mg/dl of IIM, was administered intravenously. The mean pre-treatment haemoglobin level varied from 9.6g/dl to 10.6 g/dl. The mean serum ferritin ranged from 82.4 µg/L to 161.9 µg/L and the mean transferrin saturation ranged in patients from 15.8% to 18.51%. IIM receiving patients were compared with iron sucrose receiving patients by Bhandari et al, and oral iron by Kalra et al (2015). The remaining two studies compared single IIM with multiple IIM dosages. The results are summarised in the table below.Efficacy: The increase in haemoglobin ranged between 0.9 g/dL to 6.58 g/dL from baseline measured at 8 weeks. At the same time, serum ferritin showed an increase in baseline serum ferritin from 104 µg/L to 277 µg/L and transferrin saturation (TSAT) increased in the range of 5.3%-11.4 %. All markers of iron deficiency showed significant improvement. It achieved a greater Hb response irrespective of ESA treatment in both haemodialysis dependent and non-dependent patients. In the comparison group by Bhandari et al, iron sucrose receiving patients also had similar efficacy as IIM. On the other hand, those receiving oral iron in the study by Kalra et al (2015) had much poorer efficacy as compared to IIMSafety: Severe adverse effect was seen in a total of 10 (0.6%) patients who developed hypersensitivity reaction, acute myocardial infarction and injection site-related adverse effects with hypersensitivity being the most common. 15 (0.9%) patients discontinued the treatment. 21 (1.3%) died, and the death was attributed to end-stage renal disease.ConclusionFerric derisomaltose is an effective parenteral iron therapy to treat iron deficiency when compared to oral iron therapy, however, its efficacy is very similar to other parenteral iron formulations. In terms of safety, being used as a single dose it relatively has lesser adverse events but can seldom lead to discontinuation of the drug. This drug can be recommended for iron therapy especially considering single-dose administration but more studies need to be carried out in comparison to other parenteral formulations to further clarify its efficacy in dialysis-dependent CKD patients. [Display omitted] DisclosuresAnwer: BMS / Celgene: Honoraria, Research Funding; GlaxoSmithKline: Research Funding; Janssen pharmaceutical: Honoraria, Research Funding; Allogene Therapeutics: Research Funding.

Case of Hemolytic Disease of the Newborn due to a Rare Red Cell Antibody.

Case of Hemolytic Disease of the Newborn due to a Rare Red Cell Antibody.

Efficacy and safety of ruxolitinib in ineffective erythropoiesis suppression as a pretransplantation treatment for pediatric patients with beta-thalassemia major.

Ineffective erythropoiesis (IE) is the most prominent feature of transfusion-dependent beta-thalassemia (TDT), which leads to extramedullary hemopoiesis. The rejection rate in allogeneic hematopoietic stem cell transplantation (HSCT) is high in heavily transfused patients with TDT accompanied by prominent IE. Therefore, a pretransplantation treatment bridging to HSCT is often used to reduce allosensitization and IE. Ruxolitinib is a JAK-1/JAK-2 inhibitor and has showed its efficacy in suppressing IE and the immune system. A previously published study on RUX in adult patients with TDT has revealed that this treatment significantly reduces spleen size and is well tolerated. Ten patients (5-14 years old) with TDT and an enlarged spleen were enrolled. The dose of ruxolitinib was adjusted for age: for patients<11 years: 40-100mg/m2 total daily dose and for patients>11 years: 20-30mg/m2 total daily dose. HSCT was performed in 8 of 10 patients. After the first 3 months of ruxolitinib therapy, spleen volume decreased in 9 of 10 cases by 9.1%-67.5% (M=35.4%) compared with the initial size (P=0.003). The adverse events of ruxolitinib (infectious complications, moderate thrombocytopenia, and headache) were successfully managed by reducing the dose. The outcomes of HSCT were favorable in seven of eight cases. Ruxolitinib is promising as a short-term pre-HSCT treatment for pediatric patients with TDT and pronounced IE.