Peripheral Blood Reticulocytes Research Articles

Newly discovered clouting interplay between matrix metalloproteinases structures and novel quaternary Ammonium K21: computational and in-vivo testing

Aims and objectivesTo analyze anti-MMP mode of action of Quaternary Ammonium Silane (QAS, codenamed as k21) by binding onto specific MMP site using computational molecular simulation and Anti-Sortase A (SrtA) mode of action by binding onto specific site using computational molecular simulation.Materials and methodsIn silico Molecular Dynamics (MD) was used to determine the interactions of K21 inside the pocket of the targeted protein (crystal structure of fibroblast collagenase-1 complexed to a diphenyl-ether sulphone based hydroxamic acid; PDB ID: 966C; Crystal structure of MMP-2 active site mutant in complex with APP-derived decapeptide inhibitor. MD simulations were accomplished with the Desmond package in Schrödinger Drug Discovery Suite. Blood samples (~ 0.5 mL) collected into K2EDTA were immediately transferred for further processing using the Litron MicroFlow® PLUS micronucleus analysis kit for mouse blood according to the manufacturer’s instructions. Bacterial Reverse Mutation Test of K21 Molecule was performed to evaluate K21 and any possible metabolites for their potential to induce point mutations in amino acid-requiring strains of Escherichia coli (E. coli) (WP2 uvrA (tryptophan-deficient)).ResultsMolecular Simulation depicted that K21 has a specific pocket binding on various MMPs and SrtA surfaces producing a classical clouting effect. K21 did not induce micronuclei, which are the result of chromosomal damage or damage to the mitotic apparatus, in the peripheral blood reticulocytes of male and female CD-1 mice when administered by oral gavage up to the maximum recommended dose of 2000 mg/kg. The test item, K21, was not mutagenic to Salmonella typhimurium (S. typhimurium) strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2 uvrA in the absence and presence of metabolic activation when tested up to the limit of cytotoxicity or solubility under the conditions of the test.ConclusionK21 could serve as a potent protease inhibitor maintaining the physical and biochemical properties of dental structures.

Alteration in the number, morphology, function, and metabolism of erythrocytes in high-altitude polycythemia.

Introduction: High-altitude polycythemia (HAPC) is a common chronic high-altitude disease characterized by significantly increased erythrocyte, hemoglobin (Hb), and hematocrit values and decreased arterial oxygen saturation. The mechanisms underlying HAPC development are unclear; we aimed to investigate this in an HAPC rat model. Methods: Twelve Sprague-Dawley rats were divided into control and HAPC groups. The HAPC group was exposed to hypobaric hypoxia. This HAPC model was assessed using routine blood tests and blood gas analyses. Bone marrow, peripheral blood reticulocytes (RETs), and peripheral blood erythrocyte apoptosis were measured using flow cytometry. Erythrocyte osmotic fragility (EOF) tests were conducted. Abnormal erythrocytes were counted using electron microscopy. Plasma-free hemoglobin, 5'-nucleotidase (CD73), adenosine, erythrocyte cytosolic adenosine, sphingosine-1-phosphate (S1P), and 2,3-bisphosphoglycerate (BPG) levels were measured using enzyme-linked immunosorbent assays. Erythrocyte metabolic pathway-related protein [adenosine A2B receptor (ADORA2B), erythrocyte equilibrative nucleoside transporter 1 (eENT1), sphingosine kinase 1 (SPHK1), phospho-SPHK1, bisphosphoglycerate mutase (BPGM), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] levels were assessed by Western blotting. Results: The HAPC rat model was successfully established (Hb > 210g/L). Indices of bone marrow and peripheral blood RET proportions were significantly higher in the HAPC than the control group (p = 0.04 and p < 0.001, respectively). The proportion of peripheral blood erythrocytes in early apoptosis was significantly lower in the HAPC than the control group (p < 0.001). Vesicular erythrocyte and acanthocyte proportions were significantly higher in the HAPC than the control group (p < 0.001 and p = 0.019, respectively). The EOF tests revealed that 50% erythrocyte hemolysis occurred at 4.0-4.5 and 4.5-5.0g/L NaCl in the control and HAPC groups, respectively. Plasma-free hemoglobin, CD73, adenosine, erythrocyte cytosolic adenosine, S1P, and 2,3-BPG levels and ADORA2B, eENT1, phospho-SPHK1, S1P, BPGM, and GAPDH erythrocyte expression levels (all p ≤ 0.02) were significantly higher in the HAPC than the control group. Conclusion: In model rats, an HAPC-related erythrocyte increase was associated with enhanced bone marrow hematopoietic function and reduced erythrocyte apoptosis, whereas numerous abnormal erythrocytes, increased EOF, and reduced hemolysis resistance were associated with erythrocyte metabolism. CD73/adenosine/S1P/2,3-BPG and eENT1/adenosine/BPGM/2,3-BPG metabolic pathways in erythrocytes were activated in HAPC rats, facilitating oxygen release. These findings further reveal the intrinsic HAPC mechanism and forms a basis for future development of preventive and therapeutic strategies for HAPC.

Genotoxicity and the stability of N-nitrosomorpholine activity following UVA irradiation

This study investigated N-nitrosomorpholine (NMOR) genotoxicity following UVA irradiation without metabolic activation. Following UVA irradiation, the photo treated NMOR (irradiated NMOR) was directly mutagenic, without UVA or metabolic activation, in the Ames test. The activity was relatively stable, and approximately 79% of the activity remained after 10 days of storage at 37 °C, 4 °C, or −20 °C. Micronuclei (MN) formation was observed in HaCaT cells after treatment with irradiated NMOR without metabolic activation. The action spectrum of MN formation in response to NMOR irradiation followed the NMOR absorption curve. In vivo, MN formation was observed in the peripheral blood reticulocytes of mice injected with irradiated NMOR under the inhibition of cytochrome P450-mediated metabolism of NMOR. Volatile NMOR may attach to environmental materials and be irradiated with environmental UVA light. Photoactivated NMOR-attached air pollutants could float in the air and fall onto the human body, leading to genotoxicity induced by the irradiated NMOR.

OS02-05: Genotoxicity in peripheral blood leukocytes and reticulocytes of e-waste management workers

OS02-05: Genotoxicity in peripheral blood leukocytes and reticulocytes of e-waste management workers

Recombinant α1-Microglobulin (rA1M) Protects against Hematopoietic and Renal Toxicity, Alone and in Combination with Amino Acids, in a 177Lu-DOTATATE Mouse Radiation Model.

177Lu-DOTATATE peptide receptor radionuclide therapy (PRRT) is used clinically to treat metastasized or unresectable neuroendocrine tumors (NETs). Although 177Lu-DOTATATE is mostly well tolerated in patients, bone marrow suppression and long-term renal toxicity are still side effects that should be considered. Amino acids are often used to minimize renal radiotoxicity, however, they are associated with nausea and vomiting in patients. α1-microglobulin (A1M) is an antioxidant with heme- and radical-scavenging abilities. A recombinant form (rA1M) has previously been shown to be renoprotective in preclinical models, including in PRRT-induced kidney damage. Here, we further investigated rA1M's renal protective effect in a mouse 177Lu-DOTATATE model in terms of administration route and dosing regimen and as a combined therapy with amino acids (Vamin). Moreover, we investigated the protective effect of rA1M on peripheral blood and bone marrow cells, as well as circulatory biomarkers. Intravenous (i.v.) administration of rA1M reduced albuminuria levels and circulatory levels of the oxidative stress-related protein fibroblast growth factor-21 (FGF-21). Dual injections of rA1M (i.e., at 0 and 24 h post-177Lu-DOTATATE administration) preserved bone marrow cellularity and peripheral blood reticulocytes. Administration of Vamin, alone or in combination with rA1M, did not show any protection of bone marrow cellularity or peripheral reticulocytes. In conclusion, this study suggests that rA1M, administered i.v. for two consecutive days in conjunction with 177Lu-DOTATATE, may reduce hematopoietic and kidney toxicity during PRRT with 177Lu-DOTATATE.

ASSESSMENT OF IMMATURE RETICULOCYTE FRACTION (IRF) WITH ABSOLUTE RETICULOCYTE COUNT (ARC) AS A RESPONSE MARKER IN ANTEPARTUM PATIENTS WITH NUTRITIONAL ANEMIA

Background: Peripheral blood reticulocyte count is an important index in the diagnosis, classication, and monitoring of anemic patients. After initiation of therapy, reticulocytosis develops within 3 to 7 days th th 1 and peaks (8–10%) between the 8 and 10 day . The IRF (Immature Reticulocyte Fraction) increases several days before reticulocyte count and is thus an early indicator of response to therapy. Aim: To study the response of therapy using IRF on day rd th th th 3 - 4 and day7 - 8 in antenatal patients with nutritional anemia. To correlate ARC (Absolute Reticulocyte Count) with IRF after therapy in antenatal patients with nutritional anemia. Study design: Prospective, analytical observational study. Materials and Methods: A total of 63 pregnant women with nutritional anemia were enrolled. Baseline samples for RBC indices, ARC and IRF were taken and treated with parental iron and Vit B12 injection accordingly. Baseline, 3rd - 4th and 7th – 8th day of therapy, values of hematological parameters along with ARC and IRF for predicting the response were determined. Results: The rise in mean ARC and IRF from baseline to day3/4 and day7/8 is signicantly higher in responders. There was a signicant positive correlation between IRF and ARC at baseline, day3/4 and day7/8. Conclusion: IRF and ARC increase in nutritional anemia in subjects, as early as 3-4 days post-therapy, however ARC on day 3-4 is an accurate prediction of response to therapy as evaluated on day 7-day 8 in the present study

Impact of Staining Methods and Human Factors on Accuracy of Manual Reticulocyte Enumeration.

Although peripheral blood reticulocyte enumeration reflects bone marrow functional integrity, which is important for differential diagnosis of hematological diseases, the factors affecting its accuracy have not been adequately addressed. Using 100 consecutive venous blood samples being processed with four supravital staining techniques [i.e., brilliant cresyl blue (BCB), new methylene blue (NMB), and BCB/NMB with Liu’s stain] for reticulocyte enumeration, two technologists (senior vs. junior) conducted microscopic counting. The results were compared with those obtained with an automated system (Sysmex XE-5000) that served as the standard. The aims of this study were to identify (1) the technique that gave the most reliable outcome, and (2) possible human factors (i.e., seniority, repeated counting) that may affect the counting results. Analysis showed least bias (i.e., deviation from automated counting) associated with BCB staining, followed by NMB. In addition, the senior observer exhibited a higher bias in counting compared with their junior counterpart. Repeated counting also correlated with a higher rate of bias. Nevertheless, inter-observer consistency was high (intraclass correlation coefficient >0.95) and inter-/intra-observer variations were non-significant (both p > 0.05). Our results supported the use of BCB stain for reticulocyte enumeration and the reliability of manual counting despite the involvement of human factors, which had negligible impacts on the final outcomes.

A New Murine Model of Primary Autoimmune Hemolytic Anemia (AIHA).

Loss of humoral tolerance to red blood cells (RBCs) can lead to autoimmune hemolytic anemia (AIHA), a severe, and sometimes fatal disease. Patients with AIHA present with pallor, fatigue, decreased hematocrit, and splenomegaly. While secondary AIHA is associated with lymphoproliferative disorders, infections, and more recently, as an adverse event secondary to cancer immunotherapy, the etiology of primary AIHA is unknown. Several therapeutic strategies are available; however, there are currently no licensed treatments for AIHA and few therapeutics offer treatment-free durable remission. Moreover, supportive care with RBC transfusions can be challenging as most autoantibodies are directed against ubiquitous RBC antigens; thus, virtually all RBC donor units are incompatible. Given the severity of AIHA and the lack of treatment options, understanding the cellular and molecular mechanisms that facilitate the breakdown in tolerance would provide insight into new therapeutics. Herein, we report a new murine model of primary AIHA that reflects the biology observed in patients with primary AIHA. Production of anti-erythrocyte autoantibodies correlated with sex and age, and led to RBC antigen modulation, complement fixation, and anemia, as determined by decreased hematocrit and hemoglobin values and increased reticulocytes in peripheral blood. Moreover, autoantibody-producing animals developed splenomegaly, with altered splenic architecture characterized by expanded white pulp areas and nearly diminished red pulp areas. Additional analysis suggested that compensatory extramedullary erythropoiesis occurred as there were increased frequencies of RBC progenitors detectable in the spleen. No significant correlations between AIHA onset and inflammatory status or microbiome were observed. To our knowledge, this is the first report of a murine model that replicates observations made in humans with idiopathic AIHA. Thus, this is a tractable murine model of AIHA that can serve as a platform to identify key cellular and molecular pathways that are compromised, thereby leading to autoantibody formation, as well as testing new therapeutics and management strategies.

Evaluation of the liver and blood micronucleus, and comet assay end points in a 14-day repeated-dose study with methyl carbamate and 1,3-propane sultone.

The repeated-dose liver micronucleus (RDLMN) assay is a novel method for detecting genotoxic chemicals. Two carcinogens methyl carbamate (MC) and 1,3-propane sultone (PS) were evaluated for the liver micronucleus in a 14-day repeated-dose study with Crl: CD (SD) IGS rats. Additionally, micronucleated reticulocytes (MN-RET) in peripheral blood and DNA damage (alkaline comet assay) in the liver were also assessed in the same animals. Ten groups of five male Crl: CD (SD) IGS rats were treated once daily with MC (300, 600 or 1200 mg/kg/day), PS (37.5, 75 or 150 mg/kg/day), negative control or three positive controls by oral gavage for 15 days. Blood samples were collected at 3 h after the last administration for determining MN-RET frequencies (%MN-RET), and the livers were sampled for determining the frequency of micronuclei and DNA damage. MC was negative in the comet assay, liver micronucleus assay and reticulocyte micronucleus assay, while PS was positive in all three assays. These results are consistent with the previous genotoxic findings of MC and PS. Therefore, the liver micronucleus assay can be effectively integrated into repeated-dose studies in animals. Moreover, integration of multiple genotoxicity end points into one study can reduce the number of animals, boost the experimental efficiency, and provides a comprehensive evaluation of the genotoxic potential of chemicals.

Analysis of the Outcome and Prognostic Factors of Cyclosporine A in Children with Non-severe Aplastic Anemia

To summarize the long-term efficacy of cyclosporine (CsA) in the treatment of non-severe aplastic anemia (NSAA) in children, and explore the early significant indicators. Data of 36 NSAA children in Department of Hematological Oncology, Wuhan Children's Hospital, Tongji Medical College of Huazhong University of Science and Technology from January 2013 to December 2017 were analyzed retrospectively. All the children received oral CsA immunosuppressive therapy, and CsA trough concentration was checked to maintain at the rage of 200-250 μg/L after 2 weeks. The evaluation time points were at 3, 6, 12, 18 and 24 months, and assessment items were peripheral white blood cell differential count and reticulocyte's percentage and count. The 36 NSAA cases were composed of 16 males and 20 females, whose median age was 5.46 (2.92-7.99) years old, and median follow-up time was 28.00 (10.00-38.25) months. After taking oral CsA for 24 months, the number of cumulative effective cases was 21. There were 4 cases of complete remission (CR), 17 cases of partial remission (PR), and 15 cases of non-remission (NR). The total effective rate was 58.33%, and median effect-acting time of CsA was 3.0 (0.5-10.0) months. Compared with ineffective group, neutrophil (NEU) and red blood cell (RBC) of effective group (CR+PR) began to increase significantly at the 3rd month, and hemoglobin (Hb), platelet (PLT) and white blood cell (WBC) increase significantly at the 6th month after oral CsA administration (P<0.05). Except for 2 cases who received component transfusion within 3-12 months after taking oral CsA for 3 months in effective group, the others did not need. The overall effective rate of oral CsA in children with NSAA was 58.33%. Stopping blood transfusion after the 3 months of treatment may be considered as a turning point for disease outcomes, and levels of NEU, RBC at the 3rd month and Hb, PLT, WBC at the 6th month as indicators for predicting disease prognosis.

Exchange Transfusion for Hyperbilirubinemia among Term and Near Term in NICU of a Tertiary Care Hospital of Bangladesh: Findings from a Prospective Study.

BackgroundExchange transfusion in newborns is recommended as emergency management of hyperbilirubinemia to prevent bilirubin encephalopathy and kernicterus.AimThis study aimed to determine the frequency and document common side effects of exchange transfusion and outcomes of newborns requiring exchange transfusion.Materials and methodsThis prospective study was done in the Neonatal Intensive Care Unit (NICU) of Bangabandhu Sheikh Mujib Medical University (BSMMU), Bangladesh, from January 2016 to December 2019. Information was obtained regarding maternal details, newborn demographics, and clinical status. Blood grouping and Rh typing were done for both mothers and newborns. In all newborns, pre-exchange complete blood count, peripheral blood film, Coombs test, reticulocyte count, serum bilirubin and post-exchange serum bilirubin, hemoglobin, random blood sugar, serum electrolyte, and calcium were done. G6PD level was done wherever suspected. Frequency, maternal and neonatal factors, indications, and outcomes were analyzed.ResultsAmong 839 admitted cases of unconjugated hyperbilirubinemia, 41 patients (4.9%) required exchange transfusion. Most of the babies were inborn (90.2%). Ninety-five percent of mothers received regular antenatal care; among them, 76.3% had bad obstetric history. Only 36.6% of mothers received anti-D in previous pregnancy. None had sonographic findings of hydrops. The commonest indication was Rh incompatibility (80.5%). Coombs test was positive in 58.5% of cases. Mean pre-exchange TSB was 9.44 ± 6.4, and post-exchange TSB was 4.41 ± 2.59. The commonest adverse events noted were hyperglycemia (51.2%), sepsis (19.5%), anemia requiring top-up transfusion (17.1%), and hypocalcemia (14.6%). There were no catheter-related complications. Bilirubin encephalopathy was present in 4.9% of cases. There was one mortality but not due to the procedure.ConclusionExchange transfusion was required among 4.9% of the admitted newborns with unconjugated hyperbilirubinemia. The common adverse effects were hyperglycemia and sepsis. The commonest indication was Rh incompatibility (80.5%). Overall outcome after exchange transfusion was favorable.How to cite this articleDey SK, Jahan S, Jahan I, et al. Exchange Transfusion for Hyperbilirubinemia among Term and Near Term in NICU of a Tertiary Care Hospital of Bangladesh: Findings from a Prospective Study. Euroasian J Hepato-Gastroenterol 2021;11(1):21–26.

Study of iron deficiency anemia in infants of 3 to 6 months age group and its risk factors: a cross sectional study

Background: Iron deficiency anemia in 3-6 months-old infants is often neglected. Its presence in healthy infant of less than 6 months of age is largely debated. Neglecting IDA in this critical phase of growth can lead to serious consequences. The aim and objective of the study was to estimate the frequency of IDA in 3-6 months old infants, admitted in rural tertiary care hospital and to study its risk factors.Methods: The cross-sectional study was conducted in the pediatric department of rural tertiary care hospital. 100 infants in the age group 3-6 months were included in the study. Relevant maternal and infant factors were noted. CBC with blood indices, peripheral blood smear and reticulocyte count were studied. Anemia in them was defined as hemoglobin &lt;9.5 g/dl. IDA was diagnosed based in history, examination, indices, and peripheral smear.Results: 22% of infants of 3-6 months had IDA. 40.9% of infants with IDA were low birth weight compared to 11.5% without IDA (p value&lt;0.05). All pre-terms included in the study had IDA. 16% of term, normal birth weight, exclusively breast-fed infants had IDA. Underweight and stunting were seen in 31.8 % and 30.8% of IDA infants compared to 9% and 5.4% of infants without IDA.Conclusions: IDA was quite common in infants less than 6 months and also seen in healthy, term and exclusively breast-fed babies, so universal iron supplementation before 6 months need to be considered in National Iron plus Initiative in India. Preterm, LBW babies and babies with NICU stay are at higher risk of IDA.

Maternal overnutrition before and during pregnancy induces DNA damage in male offspring: A rabbit model

Using a rabbit model, we investigated whether maternal intake of a high-fat and high-carbohydrate diet (HFCD) before and during pregnancy induces an increase in micronuclei frequency and oxidative stress in offspring during adulthood. Female rabbits received a standard diet (SD) or HFCD for two months before mating and during gestation. The offspring from both groups were nursed by foster mothers fed SD until postnatal day 35. After weaning, all the animals received SD until postnatal day 440. At postnatal day 370, the frequency of micronuclei in peripheral blood reticulocytes (MN-RETs) increased in the male offspring from HFCD-fed mothers compared with the male offspring from SD-fed mothers. Additionally, fasting serum glucose increased in the offspring from HFCD-fed mothers compared with the offspring from SD-fed mothers. At postnatal day 440, the offspring rabbits were challenged with HFCD or continued with SD for 30 days. There was an increase in MN-RET frequency in the male rabbits from HFCD-fed mothers, independent of the type of challenging diet consumed during adulthood. The challenge induced changes in serum cholesterol, LDL and HDL that were influenced by the maternal diet and offspring sex. We measured malondialdehyde in the liver of rabbits as an oxidative stress marker after diet challenge. Oxidative stress in the liver only increased in the female offspring from HFCD-fed mothers who were also challenged with this same diet. The data indicate that maternal overnutrition before and during pregnancy is able to promote different effects depending on the sex of the animals, with chromosomal instability in male offspring and oxidative stress and hypercholesterolemia in female offspring. Our data might be important in the understanding of chronic diseases that develop in adulthood due to in utero exposure to maternal diet.

Expanded Dengue Syndrome with Hemolytic Anemia: A Case Report

Introduction: Expanded Dengue Syndrome is an atypical manifestation of dengue hemorrhagic fever with compliciation of other organs such as the liver, spleen, and kidney. Haemolytic anemia is a rare complication that can occur in dengue haemorrhagic fever and the case has not been widely reported. Patient and methods: This case report will discuss a 45-year-old man who presented with severe dengue haemorrhagic fever with atypical manifestations to the other systems such as haematology, hepatology, nephrology, and gastrointestinal problems. Results: The patient was diagnosed with dengue hemorrhagic fever with the positive result of NS-1 antigen, IgG, and IgM dengue. Patient was diagnosed with severe anemia in conjunction with dengue infection. Examination of the peripheral blood smear, reticulocytes, and LDH was done with the result is haemolytic anemia. Coombs test was found negative. Transfusions of Packed Red Cells and Thrombocytes Concentrate were given as a treatment of severe anemia and thrombocytopenia. Patient recovered without further complications and was discharged home. Conclusion: The condition of hemolytic anemia in EDS is a rare condition and should be aware of. Further examination in dengue hemorrhagic fever patients with severe anemia should be considered with the presence of hemolytic anemia.

Ker-050, a Novel Inhibitor of Tgfβ Superfamily Signaling, Induces Red Blood Cell Production By Promoting Multiple Stages of Erythroid Differentiation

Diseases such as myelodysplastic syndrome (MDS) and myelofibrosis (MF) are characterized by ineffective hematopoiesis resulting in one or multiple cytopenias. Disease-causing defects occur across multiple cell lineages and stages of hematopoiesis, making development of an effective treatment for all patients challenging. Current treatment options to address anemia in these diseases target discreet stages in erythropoiesis, whereas defects leading to ineffective hematopoiesis can occur throughout the pathway. Therefore, a treatment that more globally modulates hematopoiesis has the potential to treat broad patient groups. The TGFβ superfamily plays a key role in regulating hematopoiesis; as signaling via SMAD2/3 activation results in cell quiescence, inhibiting precursors from progressing through later stages of hematopoiesis. KER-050, a modified ActRIIA ligand trap, promotes hematopoiesis through inhibition of ligands that signal though SMAD2/3, including activins and GDFs. In a Phase 1 clinical study, administration of KER-050 to healthy volunteers led to robust, rapid and sustained increases in red blood cells (RBCs), hemoglobin (HGB) and platelets, supporting an effect on the multiple stages of hematopoiesis. Here, we characterize the time course of KER-050-mediated effects on RBC production and changes in erythroid precursor cell populations in mice to characterize the mechanism of action of KER-050 on erythropoiesis. Mice treated with a single dose of a research form of KER-050 (RKER-050, 10mg/kg) had increased RBCs, HGB and hematocrit (HCT) (+8%, +9%, +7%, respectively) 12 hours after administration compared to vehicle-treated mice (VEH), and this effect was further increased on Day 7. There was also a reduction in the number of enucleated erythroid cells in the bone marrow and a parallel increase in the percent of immature reticulocytes (RET) in peripheral blood, suggesting an increased outflux of RET into circulation. This observation is consistent with RKER-050 promoting the maturation of late stage erythroid precursors leading to increases in RBCs, HGB and HCT as early as 12 hours post treatment and remaining 14 days post a single dose. In studies evaluating the effect of RKER-050 on bone marrow erythroid progenitors, a 2-fold increase in late orthochromatic erythroblasts/RETs (EryC) at Days 2 and 7 post-dose was observed. These data are consistent with RKER-050 promoting maturation and release of late-stage erythroid precursors into circulation. RKER-050 also elicited effects on early progenitors. Day 2 post-dose, there was a 2-fold increase in CFU-Es, with a 46% decrease in poly-erythrochromatic/early orthochromatic erythroid precursors (EryB) at Day 4, as compared to VEH. Day 7 post treatment, both CFU-Es and the EryB population returned to VEH levels. The increase in early progenitors appears to replenish the polychromatic erythroblasts (as shown by the return to VEH level of EryB precursors at Day 7), allowing for continued supply of maturing RETs. Consistent with this hypothesis, RKER-050-mediated changes in erythroid precursors continued to Day 14 with significantly increased early progenitor population (BFU-E +24%) and increased late stage erythroid precursors (EryB +40%, EryC 7-fold) while maintaining increased circulating RETs and RBCs. These data demonstrate that the RKER-050 treatment increases early progenitor cells which continue to mature and contribute to the overall upregulation of erythropoietic tone. Surprisingly, RKER-050 treatment resulted in a greater than 2-fold increase in serum levels of erythropoietin (Epo) at Days 4, 7 and 14. These counterintuitive results demonstrate that RKER-050 promotes erythropoiesis while at the same time increasing Epo. This effect may result in a feed-forward effect on the system and result in a sustained upregulation of erythropoietic tone. Overall, these data demonstrate that KER-050 stimulates terminal maturation of late-stage erythroid precursors, expands the early stage precursor population and progresses precursors through erythropoiesis. Additionally, KER-050 increases Epo within the milieu of elevated RBCs. The ability of KER-050 to target multiple stages along the erythropoiesis cascade makes it an appealing therapeutic candidate for diseases that cause anemia due to ineffective erythropoiesis, including MDS and MF, where defects can arise throughout the erythropoietic pathway. Disclosures Feigenson: Keros Therapeutics: Current Employment. Nathan:Keros Therapeutics: Current Employment. Materna:Keros Therapeutics: Current Employment. Gudelsky:Keros Therapeutics: Current Employment. Lema:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Tseng:Mitobridge: Current equity holder in private company; Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents &amp; Royalties. Fisher:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Seehra:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lachey:Keros Therapeutics: Current Employment, Current equity holder in publicly-traded company.

SMIM1 missense mutations exert their effect on wild type Vel expression late in erythroid differentiation.

Vel expression on erythrocytes is variable due to polymorphisms, complicating Vel typing. Weak Vel expression can be caused by mutations within SMIM1 in a heterozygous setting, suggesting a dominant negative effect of SMIM1 mutants on wild type (wt)SMIM1 expression. Here we report how SMIM1 expression is regulated during erythropoiesis, to understand its variable expression on erythrocytes. Peripheral blood reticulocytes at different stages, cultured erythroid precursors and HEK293T cells were used to investigate expression and putative competition between wtSMIM1 and mutated SMIM1 VEL*01W.01, (c.152T>A (p.Met51Lys)), VEL*01W.02 (c.152T>G (p.Met51Arg)), and VEL*01W.03 (c.161T>C (p.Leu54Pro)). Depending on the mutations in SMIM1 an effect on total and membrane expression of SMIM1 was observed in transfected HEK293T cells, but co-expression of wtSMIM1 and mutatedSMIM1 did not have an effect on wtSMIM1 membrane expression. During differentiation of donors expressing VEL*01W.01, VEL*01W.03, Vel-positive, Vel-negative (homozygote SMIM1*64_80del), and Vel-heterozygote SMIM1*64_80del primary human erythroblasts no overt defect was found in Vel expression dynamics or total SMIM1 expression levels when compared with wtSMIM1 erythroblasts. However, during enucleation, total Vel expression was significantly lower on reticulocytes of Vel-weak donors expressing heterozygote mutated SMIM1 compared to Vel-positive or Vel-heterozygote SMIM1*64_80del donors, while Vel expression on extruded nuclei was maintained. In addition, reticulocyte maturation in vivo showed further loss of Vel expression in these individuals and nearly absent on erythrocytes. These results suggest that SMIM1 mutations exert a dominant negative effect on wtSMIM1 probably by affecting SMIM1 multimerization and thereby Vel epitope presentation at the latest stages of erythroid differentiation.

A novel SPTB gene mutation in neonatal hereditary spherocytosis: A case report.

The aim of the present study was to enhance the understanding of the diagnosis and treatment of neonatal hereditary spherocytosis (HS). Gene sequencing and analysis was performed for the crucial splicing signals on the exons and introns of the 302 known pathogenic genes [including ANK1, SPTAN1, SPTA1, EPB42, SLC4A1, and SPTB] that are associated with this genetic deficiency of erythrocytes. A 26-day-old female presented with jaundice, anemia, an increased count in peripheral blood reticulocyte and spherocytes and a positive acidified glycerol hemolysis test. Gene sequencing revealed a novel mutation of c.3737delA (p.Lys1246fs) in the exon 16 of SPTB (14q23|NM_000347.5) gene in the patient and her father. The mutation was a frame-shifting mutation, which may result in the truncation of β-haemoglobin in the erythrocyte membrane can lead to loss of normal function, leading to the occurrence of diseases, including jaundice and hemolytic anemia. For neonates with jaundice and anemia, family history, erythrocyte index and peripheral blood smear findings have been indicated to contribute to the diagnosis of HS. In the current study, gene sequencing was indicated to be helpful for the diagnosis of HS. A novel mutation of SPTB gene was identified, which may be pathogenic via modulating the activity of β-spectrin in the erythrocyte membrane.

P0861THE RELATIONSHIP BETWEEN HEPCIDIN-25 AND BONE MARROW IRON IN ANEMIC, NON-DIALYSIS, CHRONIC KIDNEY DISEASE PATIENTS

Abstract Background and Aims Hepcidin-25 (Hep25) is a key known regulator of iron metabolism and its interactions with inflammation, iron stores and erythropoietic activity were involved in the pathogenesis of chronic kidney disease (CKD)-associated anemia. Therefore, our aim was to assess the determinants of serum Hep25 level in non-dialysis CKD patients. Method In this cross-sectional, single-center study, 52 subjects (56% men, 65±13 years) with CKD [estimated glomerular filtration rate, eGFR 14.5 (95%CI 16 to 25) mL/min] and anemia [hemoglobin, Hb 9.8 (95%CI 9.2 to 9.9) g/dL], not treated with erythropoiesis-stimulating agents (ESA) or iron in the previous 6 months, were enrolled. Patients with anemia of other causes than CKD, active infectious and inflammatory diseases, malignancy, severe hyperparathyroidism, transfusions during the last 3 months, and immunosuppressive therapy were excluded. The iron status was evaluated using both peripheral and central parameters. The iron stores were assessed by serum ferritin (Fer) and iron content in bone marrow macrophages (iMf, measured quantitively on a scale from 0 to 6). The iron available for erythropoiesis was assessed by transferrin saturation (TSAT) and the percentage of sideroblasts (%Sb). Anemia was further evaluated by a peripheral blood smear, erythrocytes indices and reticulocyte index. Serum Hep25 and erythropoietin (Epo) were assessed by ELISA (Bachem®, and Abcam® 119522, respectively). C-reactive protein (CRP), albumin, and parameters of kidney disease (eGFR, proteinuria) were also measured. Mann-Whitney, Kruskal-Wallis, Chi2 tests, Spearman bivariate correlation and multiple linear regression were used for statistical analysis. Results The median serum Hep25 of the whole cohort was 82.1 (95%CI 68.7 to 92.1) ng/mL. According to median Hep25, subjects were clustered in Group 1 (below median - G1) and Group 2 (above median - G2). %Sb and reticulocyte index had higher levels in G2 than in G1 [9 (95%CI 5 to 14) vs. 5 (95%CI 4 to 7) %, p=0.003 and 0.55 (95%CI 0.39 to 0.77) vs. 0.41 (95%CI 0.32 to 0.58), p=0.05, respectively], while the proportions of subjects with iMf suggestive for iron deficiency or iron overload were similar in G2 and G1 (38% vs. 50%, p=0.40, and 26% vs. 23%, p= 0.75, respectively). Peripheral blood smear, peripheral iron indices and all the other studied parameters were also alike. In bivariate analysis, Hep25 was positively associated both with indices of iron stores, i.e. Fer (rs = 0.30, p=0.03) and iMf (rs = 0.34, p=0.01) and indices of iron available for erythropoiesis, i.e. %Sb (rs = 0.55, p&amp;lt;0.001) and (marginally) with TSAT (rs = 0.26, p=0.06). Meanwhile, Hep25 was not related to serum Epo, CKD parameters or inflammation markers. In a multivariate linear regression model that explained 28% of Hep25 variation, the percentage of bone marrow sideroblasts, i.e. the tissue iron available for erythropoiesis, was the only independent determinant of Hep25: Variables entered in the first step: reticulocyte index, percentage of medullary sideroblasts (%Sb), iron content in the bone marrow macrophages (iMf), serum ferritin, and transferrin saturation Conclusion In stable patients with advanced CKD, not treated with ESA or iron, with low to moderate inflammation, serum hepcidin was related only to bone marrow iron available for erythropoiesis, suggesting that in this clinical setting the need of iron for erythropoiesis prevails over inflammation in regulation of hepcidin synthesis.

Nrf2 deficiency in mice attenuates erythropoietic stress-related macrophage hypercellularity

Erythropoiesis in the bone marrow and spleen depends on intricate interactions between the resident macrophages and erythroblasts. Our study focuses on identifying the role of nuclear factor erythroid 2-related factor 2 (Nrf2) during recovery from stress erythropoiesis. To that end, we induced stress erythropoiesis in Nrf2+/+ and Nrf2-null mice and evaluated macrophage subsets known to support erythropoiesis and erythroid cell populations. Our results confirm macrophage and erythroid hypercellularity after acute blood loss. Importantly, Nrf2 depletion results in a marked numerical reduction of F4/80+/CD169+/CD11b+ macrophages, which is more prominent under the induction of stress erythropoiesis. The observed macrophage deficiency is concomitant to a significantly impaired erythroid response to acute stress erythropoiesis in both murine bone marrow and murine spleen. Additionally, peripheral blood reticulocyte count as a response to acute blood loss is delayed in Nrf2-deficient mice compared with age-matched controls (11.0 ± 0.6% vs. 14.8 ± 0.6%, p ≤ 0.001). Interestingly, we observe macrophage hypercellularity in conjunction with erythroid hyperplasia in the bone marrow during stress erythropoiesis in Nrf2+/+ controls, with both impaired in Nrf2-/- mice. We further confirm the finding of macrophage hypercellularity in another model of erythroid hyperplasia, the transgenic sickle cell mouse, characterized by hemolytic anemia and chronic stress erythropoiesis. Our results revealed the role of Nrf2 in stress erythropoiesis in the bone marrow and that macrophage hypercellularity occurs concurrently with erythroid expansion during stress erythropoiesis. Macrophage hypercellularity is a previously underappreciated feature of stress erythropoiesis in sickle cell disease and recovery from blood loss.

Combination therapy of ginsenoside compound K and methotrexate was efficient in elimination of anaemia and reduction of disease activity in adjuvant-induced arthritis rats

Context Ginsenoside compound K (CK) has anti-inflammatory, immunoregulatory, and myelosuppressive protective effects. Methotrexate (MTX) is widely used in combination therapy for rheumatoid arthritis (RA). Objective To evaluate the effects of combination therapy of CK and MTX on anaemia and anti-arthritis in adjuvant-induced arthritis (AA) rats. Materials and methods AA was induced in rats by Complete Freund’s adjuvant, and divided into five groups (n = 10): normal, AA, CK 80 mg/kg, combination therapy (80 mg/kg CK combined with 0.5 mg/kg MTX), and MTX 0.5 mg/kg. From day 12, CK (once a day for 15 days) or MTX (once every 3 days, five times) were intragastrically administered. Results Combination therapy showed increased haemoglobin to 148.5 ± 10.1 g/L compared with AA (129.8 ± 11.7 g/L) and MTX (128.8 ± 18.4 g/L), and decreased reticulocytes in peripheral blood to 4.9 ± 1.1% compared with MTX (9.3 ± 3.3%). In combination therapy group, paw swelling decreased to 5.6 ± 4.3 mL compared with CK (9.4 ± 3.9 mL) and MTX (13.5 ± 7.4 mL), and swollen joint count decreased to 1.4 ± 0.8 compared with CK (2.1 ± 1.0) and MTX (2.4 ± 1.2) at day 24. Combination therapy showed decreased IL-6 to 25.1 ± 17.2 pg/mL compared with MTX (44.9 ± 4.8 pg/mL), and decreased IL-17 to 5.8 ± 3.9 pg/mL compared with MTX (10.7 ± 4.2 pg/mL). Conclusion The anti-anaemia effect of CK deserves further study, and CK can be a candidate effective drug for combined treatment in RA with anaemia.