Erythrocyte and reticulocyte parameters during biological monitoring of lead exposure to the body

Introduction. The most susceptible to lead is the hematopoietic system of hematopoietic organs due to lead inhibition of heme and globin synthesis and cytotoxic effect on the membrane of Mature red blood cells. The aim of study was to evaluate the informative value of the study of erythrocyte and reticulocyte parameters determined on modern hematological analyzers in patients working in contact with lead during medical and biological monitoring. Materials and methods. 45 employees of the lead battery processing plant and 30 persons of control group were examined. The level of lead in the blood was determined by atomic absorption spectrometry, δ-ALA in the urine-by the reaction of pyrol formation with acetylacetone in terms of gram of creatinine, the study of hematological parameters was performed on a Sysmex HT-2000i analyzer. Statistical processing of the results was performed using the program STATISTICA 10.0. Results. Significant changes in erythrocytic (RDW) and reticulocytic (RET, IRF, LFR, MFR, HFR, RET-He) parameters, erythropoietin in workers in contact with lead compared to the control group, changes in MCV, MCH, RDW, RET indicators in the group working in dynamics after 2 years were revealed. Associations of hematological parameters with biomarkers of exposure and effect (lead level in blood and ALA in urine) were revealed. Conclusions. Assessment of erythrocyte (MCV, MCH, RDW) and reticulocyte parameters (RET% and their distribution by maturity) in dynamics during periodic medical examinations of workers in contact with lead allows us to detect the development of hematological disorders at early stages.

Regulation of globin-heme balance in Diamond-Blackfan anemia by HSP70/GATA1

Diamond-Blackfan anemia (DBA) is a congenital erythroblastopenia that is characterized by a blockade in erythroid differentiation related to impaired ribosome biogenesis. DBA phenotype and genotype are highly heterogeneous. We have previously identified 2 in vitro erythroid cell growth phenotypes for primary CD34+ cells from DBA patients and following short hairpin RNA knockdown of RPS19, RPL5, and RPL11 expression in normal human CD34+ cells. The haploinsufficient RPS19 in vitro phenotype is less severe than that of 2 other ribosomal protein (RP) mutant genes. We further documented that proteasomal degradation of HSP70, the chaperone of GATA1, is a major contributor to the defect in erythroid proliferation, delayed erythroid differentiation, increased apoptosis, and decreased globin expression, which are all features of the RPL5 or RPL11 DBA phenotype. In the present study, we explored the hypothesis that an imbalance between globin and heme synthesis may be involved in pure red cell aplasia of DBA. We identified disequilibrium between the globin chain and the heme synthesis in erythroid cells of DBA patients. This imbalance led to accumulation of excess free heme and increased reactive oxygen species production that was more pronounced in cells of the RPL5 or RPL11 phenotype. Strikingly, rescue experiments with wild-type HSP70 restored GATA1 expression levels, increased globin synthesis thereby reducing free heme excess and resulting in decreased apoptosis of DBA erythroid cells. These results demonstrate the involvement of heme in DBA pathophysiology and a major role of HSP70 in the control of balanced heme/globin synthesis.

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

Erythropoiesis 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.

Prenatal Diagnosis of Thalassemia

Thalassemia is an individual as well as a community health problem in some countries. It causes a lifelong suffering for the affected individuals. There is no treatment other than supportive, i.e. regular transfusions and removal of iron overload from the body. Only by such continuous and expensive treatment thalassemic patients can-generally achieve nearly normal health, but the health burden of such therapy for a large number of thalassemic patients is unaffordable by the affected communities. Prevention of the births of thalassemic babies is the choice for controlling the thalassemia and has been successful in many countries. For this purpose reliable and time accurate prenatal diagnosis is a conditio sine qua non. Blood fetal sampling is safe and can be done after 16 weeks gestation, amniocentesis after 14 weeks, and even chorionic villi sampling as early as 8 weeks gestation. In vitro globin synthesis analysis applied to the fetal blood sample is very reliable to measure the rate of synthesis of the globin chains that make up the hemoglobin. The-DNA analysis of the fibroblasts obtained by amniocentesis or of the chorionic villus sample is very sensitive and specific for the diagnosis of the genetic disorder in thalassemias. By involving the prenatal diagnosis, the birth of B-homozygous thalassemia has decreased by up to 90%.

Potential new approaches to the management of the Hb Bart’s hydrops fetalis syndrome: the most severe form of α-thalassemia

The α-thalassemia trait, associated with deletions removing both α-globin genes from 1 chromosome (genotype ζ αα/ζ--), is common throughout Southeast Asia. Consequently, many pregnancies in couples of Southeast Asian origin carry a 1 in 4 risk of producing a fetus inheriting no functional α-globin genes (ζ--/ζ--), leading to hemoglobin (Hb) Bart’s hydrops fetalis syndrome (BHFS). Expression of the embryonic α-globin genes (ζ-globin) is normally limited to the early stages of primitive erythropoiesis, and so when the ζ-globin genes are silenced, at ∼6 weeks of gestation, there should be no α-like globin chains to pair with the fetal γ-globin chains of Hb, which consequently form nonfunctional tetramers (γ4) known as Hb Bart’s. When deletions leave the ζ-globin gene intact, a low level of ζ-globin gene expression continues in definitive erythroid cells, producing small amounts of Hb Portland (ζ2γ2), a functional form of Hb that allows the fetus to survive up to the second or third trimester. Untreated, all affected individuals die at these stages of development. Prevention is therefore of paramount importance. With improvements in early diagnosis, intrauterine transfusion, and advanced perinatal care, there are now a small number of individuals with BHFS who have survived, with variable outcomes. A deeper understanding of the mechanism underlying the switch from ζ- to α-globin expression could enable persistence or reactivation of embryonic globin synthesis in definitive cells, thereby providing new therapeutic options for such patients.

Protective Role of Histidine Supplementation Against Oxidative Stress Damage in the Management of Anemia of Chronic Kidney Disease

Anemia is a major health condition associated with chronic kidney disease (CKD). A key underlying cause of this disorder is iron deficiency. Although intravenous iron treatment can be beneficial in correcting CKD-associated anemia, surplus iron can be detrimental and cause complications. Excessive generation of reactive oxygen species (ROS), particularly by mitochondria, leads to tissue oxidation and damage to DNA, proteins, and lipids. Oxidative stress increase in CKD has been further implicated in the pathogenesis of vascular calcification. Iron supplementation leads to the availability of excess free iron that is toxic and generates ROS that is linked, in turn, to inflammation, endothelial dysfunction, and cardiovascular disease. Histidine is indispensable to uremic patients because of the tendency toward negative plasma histidine levels. Histidine-deficient diets predispose healthy subjects to anemia and accentuate anemia in chronic uremic patients. Histidine is essential in globin synthesis and erythropoiesis and has also been implicated in the enhancement of iron absorption from human diets. Studies have found that L-histidine exhibits antioxidant capabilities, such as scavenging free radicals and chelating divalent metal ions, hence the advocacy for its use in improving oxidative stress in CKD. The current review advances and discusses evidence for iron-induced toxicity in CKD and the mechanisms by which histidine exerts cytoprotective functions.

BAP1 deubiquitinase is a potent repressor of fetal hemoglobin biosynthesis

SummaryHuman globin gene production transcriptionally “switches” from fetal to adult synthesis shortly after birth, and is controlled by macromolecular complexes that enhance or suppress transcription by cis-elements scattered throughout the locus. The DRED repressor is recruited to the ε- and γ-globin promoters by the orphan nuclear receptors TR2 (NR2C1) and TR4 (NR2C2) to engender their silencing in adult erythroid cells. Here we found that nuclear receptor corepressor-1 (NCoR1) is a critical component of DRED that acts as a scaffold to unite the DNA binding and epigenetic enzyme components (e.g. DNMT1 and LSD1) that elicit DRED function. We also describe a potent new regulator of γ-globin repression: the deubiquitinase BAP1 is a component of the repressor complex whose activity maintains NCoR1 at sites in the β-globin locus, and BAP1 inhibition in erythroid cells massively induces γ-globin synthesis. These data provide new mechanistic insights through the discovery of novel epigenetic enzymes that mediate γ-globin gene repression.

Delayed Globin Synthesis Leads to Excessive Heme and the Macrocytic Anemia of Diamond Blackfan Anemia and del(5q) Myelodysplastic Syndrome

Thirty percent of Diamond Blackfan anemia (DBA) cases result from haploinsufficiency of ribosomal protein S19 and ~40% from haploinsufficiencies of 15 other ribosomal proteins. The macrocytic anemia of myelodysplasia with deletion of chromosome 5q (del(5q)MDS), which results from the acquisition of RPS14 haploinsufficiency, has a similar clinical phenotype. Although these mutations disrupt ribosome assembly and impair protein translation, how this causes macrocytic anemia remains uncertain and controversial. Since 95% of the protein content of red cells is globin, we hypothesized that any germline or somatic mutation that slows protein synthesis would impair globin production relative to heme production. This is because the synthesis of heme, a chemical chelate, depends on only small amounts of protein (enzymes) and the rate limiting enzyme, ALAS2, is an early GATA1 target. Studies of marrow cells from patients with DBA and del(5q) MDS show that heme synthesis indeed progresses normally, while globin synthesis is delayed. This results in excess heme in CFU-E/proerythroblasts, excessive ROS and cell death (Sci Transl Med 8:338RA67, 2016). Similar results are seen in a murine model of heme excess (Flvcr1 -deletion) (J Clin Invest 125:4681, 2015) and murine models of DBA. As slowing heme synthesis improves the coordination of heme and globin and improves red cell production, a phase 2 study is underway (PI Bart Scott) to determine the efficacy of aggressive iron chelation to slow heme synthesis in patients with very low to intermediate risk MDS and anemia. More recently, we have quantitated the cell surface expression of CD71, CD44 and Ter119 on individual murine erythroid cells from normal, Flvcr1 -deleted mice with macrocytic anemia, and erythropoietin-treated mice. We then barcoded and assessed the cell's total transcriptome. By linking these datasets, we uncovered a GATA1-heme autoregulatory loop which regulates normal erythropoiesis and contributes to the failed erythropoiesis of ribosomal protein haploinsufficiency. We show that heme normally upregulates ribosome protein transcription in early erythroid cells. Thus, in addition to increasing globin transcription and translation (via Bach1 and HRI), heme assures adequate ribosomes for globin synthesis. In later erythroid cells, heme decreases GATA1, GATA1 target genes and mitotic spindle gene expression, assuring that red cell differentiation appropriately terminates and cell division ceases. In human marrow CD36+GlyA- or CD36+GlyA+ cells, these changes occur within 15 minutes of inducing endogenous heme synthesis with ALA (bypasses ALAS2) and iron. As excess heme would increase ROS, increase ribosomal protein imbalance to intensify P53 expression, prematurely lower GATA1, and impede mitosis, our data explain the ineffective (early termination of) erythropoiesis in DBA and del(5q) MDS, help explain why these anemias are macrocytic, and reconcile the disparate observations of others. Disclosures No relevant conflicts of interest to declare.