Expression of microRNA-155 in thalassemic erythropoiesis.

Background: Ineffective erythropoiesis (IE) is the primary cause of anemia and associated pathologies in ß-thalassemia. The characterization of IE is imbalance of erythroid proliferation and differentiation, resulting in increased erythroblast proliferation that fails to differentiate and gives rise to enucleate RBCs. MicroRNAs (miRs) are known to play important roles in hematopoiesis. miR-155 is a multifunctional molecule involved in both normal and pathological hematopoiesis, and its upregulation is observed in patients with ß-thalassemia/HbE. However, the expression and function of miR-155, especially in ß-thalassemia, have not yet been explored. Methods: To study miR-155 expression in thalassemia, erythroblast subpopulations, CD45-CD71+Ter-119+ and CD45-CD71-Ter-119+ were collected from ß IVSII-654 thalassemic bone marrow. Additionally, a two-phase culture of mouse bone marrow erythroid progenitor cells was performed. Expression of miR-155 and predicted mRNA target genes, c-myc, bach-1 and pu-1, were determined by quantitative reverse transcription (qRT)-polymerase chain reaction (PCR) and normalized to small nucleolar RNA (snoRNA) 202 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. To investigate the effect of miR-155 expression, erythroblasts were transfected with miR-inhibitor and -mimic in order to elevate and eliminate miR-155 expression, respectively. Erythroid cell differentiation was evaluated by Wright-Giemsa staining and flow cytometry. Results: miR-155 was upregulated, both in vivo and in vitro, during erythropoiesis in ß-thalassemic mice. Our study revealed that gain- and loss of function of miR-155 were involved in erythroid proliferation and differentiation, and augmented proliferation and differentiation of thalassemic mouse erythroblasts may be associated with miR-155 upregulation. miR-155 upregulation in ß-thalassemic mice significantly increased the percentage of basophilic and polychromatic erythroblasts. Conversely, a significant decrease in percentage of basophilic and polychromatic erythroblasts was observed in ß-thalassemic mice transfected with anti-miR-155 inhibitor. We also examined the mRNA targets (c-myc, bach-1 and pu-1) of miR-155, which indicated that c-myc is a valid target gene of miR-155 that regulates erythroid differentiation. Conclusion: miR-155 regulates IE in ß-thalassemia via c-myc expression controlling erythroblast proliferation and differentiation.

Transcriptional Repressor BCL11A in Erythroid Cells.

BCL11A, a zinc finger repressor, is a stage-specific transcription factor that controls the switch from fetal (HbF, α2γ2) to adult (HbA, α2ß2) hemoglobin in erythroid cells. While BCL11A was known as a factor critical for B-lymphoid cell development, its relationship to erythroid cells and HbF arose through genome-wide association studies (GWAS). Subsequent work validated its role as a silencer of γ-globin gene expression in cultured cells and mice. Erythroid-specific loss of BCL11A rescues the phenotype of engineered sickle cell disease (SCD) mice, thereby suggesting that downregulation of BCL11A expression might be beneficial in patients with SCD and ß-thalassemia. Common genetic variation in GWAS resides in an erythroid-specific enhancer within the BCL11A gene that is required for its own expression. CRISPR/Cas9 gene editing of the enhancer revealed a GATA-binding site that confers a large portion of its regulatory function. Disruption of the GATA site leads to robust HbF reactivation. Advancement of a guide RNA targeting the GATA-binding site in clinical trials has recently led to approval of first-in-man use of ex vivo CRISPR editing of hematopoietic stem/progenitor cells (HSPCs) as therapy of SCD and ß-thalassemia. Future challenges include expanding access and infrastructure for delivery of genetic therapy to eligible patients, reducing potential toxicity and costs, exploring prospects for in vivo targeting of hematopoietic stem cells (HSCs), and developing small molecule drugs that impair function of BCL11A protein as an alternative option.

Therapeutic Relevance of Inducing Autophagy in ß-Thalassemia.

The ß-thalassemias are inherited genetic disorders affecting the hematopoietic system. In ß-thalassemias, more than 350 mutations of the adult ß-globin gene cause the low or absent production of adult hemoglobin (HbA). A clinical parameter affecting the physiology of erythroid cells is the excess of free α-globin. Possible experimental strategies for a reduction in excess free α-globin chains in ß-thalassemia are CRISPR-Cas9-based genome editing of the ß-globin gene, forcing "de novo" HbA production and fetal hemoglobin (HbF) induction. In addition, a reduction in excess free α-globin chains in ß-thalassemia can be achieved by induction of the autophagic process. This process is regulated by the Unc-51-like kinase 1 (Ulk1) gene. The interplay with the PI3K/Akt/TOR pathway, with the activity of the α-globin stabilizing protein (AHSP) and the involvement of microRNAs in autophagy and Ulk1 gene expression, is presented and discussed in the context of identifying novel biomarkers and potential therapeutic targets for ß-thalassemia.

Elevating fetal hemoglobin: recently discovered regulators and mechanisms.

ABSTRACT: It has been known for over half a century that throughout ontogeny, humans produce different forms of hemoglobin, a tetramer of α- and ß-like hemoglobin chains. The switch from fetal to adult hemoglobin occurs around the time of birth when erythropoiesis shifts from the fetal liver to the bone marrow. Naturally, diseases caused by defective adult ß-globin genes, such as sickle cell disease and ß-thalassemia, manifest themselves as the production of fetal hemoglobin fades. Reversal of this developmental switch has been a major goal to treat these diseases and has been a driving force to understand its underlying molecular biology. Several review articles have illustrated the long and at times arduous paths that led to the discovery of the first transcriptional regulators involved in this process. Here, we survey recent developments spurred by the discovery of CRISPR tools that enabled for the first time high-throughput genetic screens for new molecules that impact the fetal-to-adult hemoglobin switch. Numerous opportunities for therapeutic intervention have thus come to light, offering hope for effective pharmacologic intervention for patients for whom gene therapy is out of reach.

Proteomic profiling of circulating ß-thalassaemia/haemoglobin E extra-cellular vesicles reveals that association with immunoglobulin induces membrane vesiculation.

Splenectomised ß-thalassaemia/haemoglobin E (HbE) patients have increased levels of circulating microparticles or medium extra-cellular vesicles (mEVs). The splenectomised mEVs play important roles in thromboembolic complications in patients since they can induce platelet activation and endothelial cell dysfunction. However, a comprehensive understanding of the mechanism of mEV generation in thalassaemia disease has still not been reached. Thalassaemic mEVs are hypothesised to be generated from cellular oxidative stress in red blood cells (RBCs) and platelets. Therefore, a proteomic analysis of mEVs from splenectomised and non-splenectomised ß-thalassaemia/HbE patients was performed by liquid chromatography with tandem mass spectrometry. A total of 171 proteins were identified among mEVs. Interestingly, 72 proteins were uniquely found in splenectomised mEVs including immunoglobulin subunits and cytoskeleton proteins. Immunoglobulin G (IgG)-bearing mEVs in splenectomised patients were significantly increased. Furthermore, complement C1q was detected in both mEVs with IgG binding and mEVs without IgG binding. Interestingly, the percentage of mEVs generated from RBCs with IgG binding was approximately 15-20 times higher than the percentage of RBCs binding with IgG. This suggested that the vesiculation of thalassaemia mEVs could be a mechanism of RBCs to eliminate membrane patches harbouring immune complex and may consequently prevent cells from phagocytosis and lysis.

GDF15 linked to maternal risk of nausea and vomiting during pregnancy.

GDF15, a hormone acting on the brainstem, has been implicated in the nausea and vomiting of pregnancy, including its most severe form, hyperemesis gravidarum (HG), but a full mechanistic understanding is lacking1-4. Here we report that fetal production of GDF15 and maternal sensitivity to it both contribute substantially to the risk of HG. We confirmed that higher GDF15 levels in maternal blood are associated with vomiting in pregnancy and HG. Using mass spectrometry to detect a naturally labelled GDF15 variant, we demonstrate that the vast majority of GDF15 in the maternal plasma is derived from the feto-placental unit. By studying carriers of rare and common genetic variants, we found that low levels of GDF15 in the non-pregnant state increase the risk of developing HG. Conversely, women with ß-thalassaemia, a condition in which GDF15 levels are chronically high5, report very low levels of nausea and vomiting of pregnancy. In mice, the acute food intake response to a bolus of GDF15 is influenced bi-directionally by prior levels of circulating GDF15 in a manner suggesting that this system is susceptible to desensitization. Our findings support a putative causal role for fetally derived GDF15 in the nausea and vomiting of human pregnancy, with maternal sensitivity, at least partly determined by prepregnancy exposure to the hormone, being a major influence on its severity. They also suggest mechanism-based approaches to the treatment and prevention of HG.

Decrease in α-Globin and Increase in the Autophagy-Activating Kinase ULK1 mRNA in Erythroid Precursors from ß-Thalassemia Patients Treated with Sirolimus.

The ß-thalassemias are hereditary monogenic diseases characterized by a low or absent production of adult hemoglobin and excess in the content of α-globin. This excess is cytotoxic for the erythroid cells and responsible for the ß-thalassemia-associated ineffective erythropoiesis. Therefore, the decrease in excess α-globin is a relevant clinical effect for these patients and can be realized through the induction of fetal hemoglobin, autophagy, or both. The in vivo effects of sirolimus (rapamycin) and analogs on the induction of fetal hemoglobin (HbF) are of key importance for therapeutic protocols in a variety of hemoglobinopathies, including ß-thalassemias. In this research communication, we report data showing that a decrease in autophagy-associated p62 protein, increased expression of ULK-1, and reduction in excess α-globin are occurring in erythroid precursors (ErPCs) stimulated in vitro with low dosages of sirolimus. In addition, increased ULK-1 mRNA content and a decrease in α-globin content were found in ErPCs isolated from ß-thalassemia patients recruited for the NCT03877809 clinical trial and treated with 0.5-2 mg/day sirolimus. Our data support the concept that autophagy, ULK1 expression, and α-globin chain reduction should be considered important endpoints in sirolimus-based clinical trials for ß-thalassemias.

Generation of human induced pluripotent stem cell line (MUi033-A) from a male with homozygous for Hemoglobin E.

Hemoglobin E (HbE), a common variant in Southeast Asian populations, results from a G to A substitution at codon 26 of the HBB gene, causing abnormal Hb and mild ß-thalassemia-like symptoms. Here, we derived an induced pluripotent stem cell (iPSC) line, named MUi033-A, from a male homozygous for HbE. The iPSC line demonstrates a normal karyotype and embryonic stem cell-like properties including pluripotency gene expression, and tri-lineage differentiation potential. This iPSC resource holds the potential for investigating gene therapy targeting HbE mutation.

CRISPR/Cas-based gene editing in therapeutic strategies for beta-thalassemia.

Beta-thalassemia (ß-thalassemia) is an autosomal recessive disorder caused by point mutations, insertions, and deletions in the HBB gene cluster, resulting in the underproduction of ß-globin chains. The most severe type may demonstrate complications including massive hepatosplenomegaly, bone deformities, and severe growth retardation in children. Treatments for ß-thalassemia include blood transfusion, splenectomy, and allogeneic hematopoietic stem cell transplantation (HSCT). However, long-term blood transfusions require regular iron removal therapy. For allogeneic HSCT, human lymphocyte antigen (HLA)-matched donors are rarely available, and acute graft-versus-host disease (GVHD) may occur after the transplantation. Thus, these conventional treatments are facing significant challenges. In recent years, with the advent and advancement of CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) gene editing technology, precise genome editing has achieved encouraging successes in basic and clinical studies for treating various genetic disorders, including ß-thalassemia. Target gene-edited autogeneic HSCT helps patients avoid graft rejection and GVHD, making it a promising curative therapy for transfusion-dependent ß-thalassemia (TDT). In this review, we introduce the development and mechanisms of CRISPR/Cas9. Recent advances on feasible strategies of CRISPR/Cas9 targeting three globin genes (HBB, HBG, and HBA) and targeting cell selections for ß-thalassemia therapy are highlighted. Current CRISPR-based clinical trials in the treatment of ß-thalassemia are summarized, which are focused on γ-globin reactivation and fetal hemoglobin reproduction in hematopoietic stem cells. Lastly, the applications of other promising CRISPR-based technologies, such as base editing and prime editing, in treating ß-thalassemia and the limitations of the CRISPR/Cas system in therapeutic applications are discussed.

BCL11A-targeted γ-globin gene induction by triterpenoid glycosides of Fagonia indica: A preclinical scientific validation of indigenous herb for the treatment of ß-hemoglobinopathies.

Pharmacological induction of fetal hemoglobin has proven to be a promising therapeutic intervention in ß-hemoglobinopathies by reducing the globin chain imbalance and inhibiting sickle cell polymerization. Fagonia indica has shown therapeutic relevance to ß-thalassemia. Therefore, we study the ethnopharmacological potential of Fagonia indica and its biomarker compounds for their HbF induction ability for the treatment of ß-thalassemia. Here, we identify, compound 8 (triterpenoid glycosides) of F. indica. as a prominent HbF inducer in-vitro and in-vivo. Compound 8 showed potent erythroid differentiation, enhanced cellular proliferation, ample accumulation of total hemoglobin, and a strong notion of γ-globin gene expression in K562 cultures. Compound 8 treatment also revealed strong induction of erythroid differentiation and fetal hemoglobin mRNA and protein in adult erythroid precursor cells. This induction was associated with simultaneous downregulation of BCL11A and SOX6, and overexpression of the GATA-1 gene, suggesting a compound 8-mediated partial mechanism involved in the reactivation of fetal-like globin genes. The in vivo study with compound 8 (10 mg/kg) in ß-YAC mice resulted in significant HbF synthesis demonstrated by the enhanced level of F-cells (84.14 %) and an 8.85-fold increase in the γ-globin gene. Overall, the study identifies compound 8 as a new HbF-inducing entity and provides an early "proof-of-concept" to enable the initiation of preclinical and clinical studies in the development of this HbF-inducing agent for ß-thalassemia.

Up-regulation of microRNA 101-3p during erythropoiesis in ß-thalassemia/HbE.

Ineffective erythropoiesis is the main cause of anemia in ß-thalassemia. The crucial hallmark of ineffective erythropoiesis is the high proliferation of erythroblast. microRNA (miR/miRNA) involves several biological processes, including cell proliferation and erythropoiesis. miR-101 was widely studied and associated with proliferation in several types of cancer. However, the miR-101-3p has not been studied in ß-thalassemia/HbE. Therefore, this study aims to investigate the expression of miR-101-3p during erythropoiesis in ß-thalassemia/HbE. The results showed that miR-101-3p was upregulated in the erythroblast of ß-thalassemia/HbE patients on day 7, indicating that miR-101-3p may be involved with high proliferation in ß-thalassemia/HbE. Therefore, the mRNA targets of miR-101-3p including Rac1, SUB1, TET2, and TRIM44 were investigated to determine the mechanisms involved with high proliferation of ß-thalassemia/HbE erythroblasts. Rac1 expression was significantly reduced at day 11 in severe ß-thalassemia/HbE compared to normal controls and mild ß-thalassemia/HbE. SUB1 gene expression was significantly lower in severe ß-thalassemia/HbE compared to normal controls at day 9 of culture. For TET2 and TRIM44 expression, a significant difference was not observed among normal and ß-thalassemia/HbE. However, the high expression of miR-101-3p at day 7 and these target genes was not correlated, suggesting that this miRNA may regulate ineffective erythropoiesis in ß-thalassemia/HbE via other target genes.

Precision Editing as a Therapeutic Approach for ß-Hemoglobinopathies.

Beta-hemoglobinopathies are the most common genetic disorders worldwide, caused by a wide spectrum of mutations in the ß-globin locus, and associated with morbidity and early mortality in case of patient non-adherence to supportive treatment. Allogeneic transplantation of hematopoietic stem cells (allo-HSCT) used to be the only curative option, although the indispensable need for an HLA-matched donor markedly restricted its universal application. The evolution of gene therapy approaches made possible the ex vivo delivery of a therapeutic ß- or γ- globin gene into patient-derived hematopoietic stem cells followed by the transplantation of corrected cells into myeloablated patients, having led to high rates of transfusion independence (thalassemia) or complete resolution of painful crises (sickle cell disease-SCD). Hereditary persistence of fetal hemoglobin (HPFH), a syndrome characterized by increased γ-globin levels, when co-inherited with ß-thalassemia or SCD, converts hemoglobinopathies to a benign condition with mild clinical phenotype. The rapid development of precise genome editing tools (ZFN, TALENs, CRISPR/Cas9) over the last decade has allowed the targeted introduction of mutations, resulting in disease-modifying outcomes. In this context, genome editing tools have successfully been used for the introduction of HPFH-like mutations both in HBG1/HBG2 promoters or/and in the erythroid enhancer of BCL11A to increase HbF expression as an alternative curative approach for ß-hemoglobinopathies. The current investigation of new HbF modulators, such as ZBTB7A, KLF-1, SOX6, and ZNF410, further expands the range of possible genome editing targets. Importantly, genome editing approaches have recently reached clinical translation in trials investigating HbF reactivation in both SCD and thalassemic patients. Showing promising outcomes, these approaches are yet to be confirmed in long-term follow-up studies.

Novel potential therapeutics to modify iron metabolism and red cell synthesis in diseases associated with defective erythropoiesis.

Under normal conditions, iron metabolism is carefully regulated to sustain normal cellular functions and the production of hemoglobin in erythroid cells. Perturbation to the erythropoiesis-iron metabolism axis can result in iron imbalances and cause anemia or organ toxicity. Various congenital and acquired diseases associated with abnormal red cell production are characterized by aberrant iron absorption. Several recent studies have shown that improvements in red blood cell production also ameliorate iron metabolism and vice versa. Many therapeutics are now under development with the potential to improve a variety of hematologic diseases, from ß-thalassemia and iron-refractory iron deficiency anemia to anemia of inflammation and polycythemia vera. This review summarizes selected mechanisms related to red cell production and iron metabolism and describes potential therapeutics and their current uses. We also consider the potential application of the discussed therapeutics on various diseases, alone or in combination. The vast repertoire of drugs under development offers new opportunities to improve the clinical care of patients suffering from congenital or acquired red blood cell disorders with limited or no treatment options.

Fetal factors disrupt placental and maternal iron homeostasis in murine ß-thalassemia.

Pregnancy rates in ß-thalassemia are increasing but the risk of complications is higher; thus, better understanding of maternal and fetal iron homeostasis in this disorder is needed. HbbTh3/+ (Th3/+) mice model human ß-thalassemia. Both the murine and human diseases are characterized by low hepcidin, high iron absorption, and tissue iron overload, with concurrent anemia. We hypothesized that disordered iron metabolism in pregnant Th3/+ mice would negatively affect their unborn offspring. The experimental design included these groups: wild-type (WT) dams carrying WT fetuses (WT1); WT dams carrying WT and Th3/+ fetuses (WT2); Th3/+ dams carrying WT and Th3/+ fetuses (Th3/+); and age-matched, nonpregnant adult females. Serum hepcidin was low, and mobilization of splenic and hepatic storage iron was enhanced in all 3 groups of experimental dams. Intestinal 59Fe absorption was lower in Th3/+ dams (as compared with WT1/2 dams) but splenic 59Fe uptake was higher. Th3/+ dams had hyperferremia, which led to fetal and placenta iron loading, fetal growth restriction, and placentomegaly. Notably, Th3/+ dams loaded Th3/+ and WT fetuses, with the latter situation more closely mirroring human circumstances when mothers with thalassemia have relatively unaffected (thalassemia trait) offspring. Iron-related oxidative stress likely contributed to fetal growth impairment; enhanced placental erythropoiesis is a probable cause of placental enlargement. Moreover, high fetal liver iron transactivated Hamp; fetal hepcidin downregulated placental ferroportin expression, limiting placental iron flux and thus mitigating fetal iron loading. Whether gestational iron loading occurs in human thalassemic pregnancy, when blood transfusion can further elevate serum iron, is worth consideration.

Single-cell profiling of ineffective erythropoiesis in a mouse model of ß-thalassaemia intermedia.

Beta-thalassaemia is an inherited haemoglobin disorder characterised by ineffective erythropoiesis (IE). The detailed pathogenesis of IE remains unclear. In this study, we used single-cell RNA sequencing (scRNA-seq) to examine IE in Th3/+ ß-thalassaemic mice. The results showed that the erythroid group was remarkably expanded, and genes involved in biological processes such as iron metabolism, haeme synthesis, protein folding, and response to heat were significantly upregulated from erythroid progenitors to reticulocytes in ß-thalassaemic mice. In particular, we identified a unique cell population close to reticulocytes, named ThReticulocytes, characterised by a high level of heat shock protein 70 (Hsp70) expression and dysregulation of iron metabolism and haeme synthesis signalling. Treatment of ß-thalassaemic mice with the haeme oxygenase inhibitor tin-mesoporphyrin effectively improved the iron disorder and IE, and the ThReticulocyte population and Hsp70 expression were significantly suppressed. This study revealed in detail the progression of IE at the single-cell level and possibly provided clues to find therapeutic targets in thalassaemia.

Discovery-Based Proteomics Identify Skeletal Muscle Mitochondrial Alterations as an Early Metabolic Defect in a Mouse Model of ß-Thalassemia.

Although metabolic complications are common in thalassemia patients, there is still an unmet need to better understand underlying mechanisms. We used unbiased global proteomics to reveal molecular differences between the th3/+ mouse model of thalassemia and wild-type control animals focusing on skeletal muscles at 8 weeks of age. Our data point toward a significantly impaired mitochondrial oxidative phosphorylation. Furthermore, we observed a shift from oxidative fibre types toward more glycolytic fibre types in these animals, which was further supported by larger fibre-type cross-sectional areas in the more oxidative type fibres (type I/type IIa/type IIax hybrid). We also observed an increase in capillary density in th3/+ mice, indicative of a compensatory response. Western blotting for mitochondrial oxidative phosphorylation complex proteins and PCR analysis of mitochondrial genes indicated reduced mitochondrial content in the skeletal muscle but not the hearts of th3/+ mice. The phenotypic manifestation of these alterations was a small but significant reduction in glucose handling capacity. Overall, this study identified many important alterations in the proteome of th3/+ mice, amongst which mitochondrial defects leading to skeletal muscle remodelling and metabolic dysfunction were paramount.

Oleic acid prevents erythrocyte death by preserving haemoglobin and erythrocyte membrane proteins.

Haemolysis of erythrocytes upon exposure to haemato-toxic phenylhydrazine (PHZ), makes it an experimental model of anaemia and a partial model of ß-thalassaemia, where oxidative stress (OS) was identified as principal causative factor. Oleic acid (OA) was evidenced to ameliorate such stress with antioxidative potential. Erythrocytes were incubated in vitro using 1 mM PHZ, 0.06 nM OA. Erythrocyte membrane protein densities and haemoglobin (Hb) status were examined. Any interaction of Hb with PHZ/OA was checked by calorimetric and spectroscopic analysis using pure molecules. Occurrence of erythrocyte apoptosis and involvement of free iron in all groups were evaluated. PHZ exposure to erythrocytes results in OS with subsequent apoptosis as evidenced from increased lipid peroxidation and translocation of phosphatidylserine in outer membrane. Preservations of erythrocyte cytoskeletal architecture and membrane bound enzyme activity were found in presence of OA. Moreover, both heme and globin of Hb was examined to be conserved by OA. Presence of OA, impeded apoptosis also, possibly by thwarting Hb breakdown followed by free iron release and consequent free radical generation. Additionally, direct sequential binding of OA with PHZ endorsed another protective mechanism of OA toward erythrocytes. OA affords protection to erythrocytes by conserving its major components and prevents haemolysis which project OA as a haemato-protective agent. Apart from combating PHZ toxicity, anti-apoptotic action of OA strongly suggests its usage in anaemia and ß-thalassaemia patients to curb irreversible erythrocyte breakdown. This research strongly recommends OA in pure form or from dietary sources as a therapeutic against haemolytic disorders.

The rs368698783 (G>A) Polymorphism Affecting LYAR Binding to the Aγ-Globin Gene Is Associated with High Fetal Hemoglobin (HbF) in ß-Thalassemia Erythroid Precursor Cells Treated with HbF Inducers

The human homologue of mouse Ly-1 antibody reactive clone protein (LYAR) is a putative novel regulator of γ-globin gene transcription. The LYAR DNA-binding motif (5'-GGTTAT-3') is located within the 5'-UTR of the Aγ-globin gene. The LYAR rs368698783 (G>A) polymorphism is present in ß-thalassemia patients and decreases the LYAR binding efficiency to the Aγ-globin gene. The objective of this study was to stratify ß-thalassemia patients with respect to the rs368698783 (G>A) polymorphism and to verify whether their erythroid precursor cells (ErPCs) differentially respond in vitro to selected fetal hemoglobin (HbF) inducers. The rs368698783 (G>A) polymorphism was detected by DNA sequencing, hemoglobin production by HPLC, and accumulation of globin mRNAs by RT-qPCR. We found that the LYAR rs368698783 (G>A) polymorphism is associated with high basal and induced production of fetal hemoglobin in ß-thalassemia patients. The most striking association was found using rapamycin as an HbF inducer. The results presented here could be considered important not only for basic biomedicine but also in applied translational research for precision medicine in personalized therapy of ß-thalassemia. Accordingly, our data suggest that the rs368698783 polymorphism might be considered among the parameters useful to recruit patients with the highest probability of responding to in vivo hydroxyurea (HU) treatment.

Liver fibrosis in young patients with transfusion-dependent thalassaemia (TDT), understanding the role of steatosis.

Using transient elastography Padeniya et al. detected steatosis and fibrosis in a cohort of young heavily iron overloaded patients with transfusion-dependent thalassaemia; steatosis was associated only with increasing body mass index, but not with iron overload and diabetes. Recently, great efforts have been devoted to eliminating or reducing iron overload and hepatitis C infection, which are well-recognised causes of liver damage. Thus, haematologists should be aware that steatosis and probably more complex metabolism alterations may be encountered very early in these patients and could be responsible for further liver damage. Commentary on: Padeniya et al. The association between steatosis and liver damage in transfusion-dependent beta thalassaemia patients. Br J Haematol 2023;200:517-523.

Isolation and in vitro Expansion of Regulatory T Cells from ß-Thalassemia Major Kids and in vitro Evaluation of Their Plasticity and Inhibitory Effects in the Presence of Pro-Inflammatory Cytokines.

INTRODUCTION: Graft-versus-host disease (GvHD) is a life-threatening syndrome commonly associated with hematopoietic stem cell transplantation (HSCT). Preventing the incidence of GVHD after HSCT along with minimizing long-term immunosuppression is currently under investigation with regulatory T cells (Tregs). As Tregs are a low-frequency population and the yield of all memory Tregs is not sufficient for clinical application, an initial Treg expansion is essential. METHODS: Thirty milliliters of peripheral blood from the ß-thalassemia major (beta-TM) patients and healthy controls were obtained and Tregs were isolated using MACS. Isolated cells were cultured in the presence of rapamycin and rIL-2 followed by activated with anti-CD3/CD28-coated beads. To evaluate Treg plasticity, expanded Tregs were cultured in a medium containing IL1ß, IL6, TGFß, and IL2, with or without 500 nM rapamycin for 72 h. To assess the functional properties of Tregs, CFSE dilution assays were performed to evaluate the ability of in vivo expanded Tregs from beta-TM patients. Statistical analysis was performed using paired t-test and independent t-test, with the aid of SPSS version 12.0. p-value ≤0.05 was considered significant. RESULTS: The percentage of Tregs isolated from the control group was significantly higher than the Tregs isolated from patients (p-value = 0.01), which is probably due to the iron overload in beta-TM patients as a result of continuous blood transfusion. Also, the percentage of Tregs after 5 days of expansion had a significant increase in both groups compared to before expansion (p-value = 0.03). Our results also showed that the expansion of Tregs after 72 h in the presence of inflammatory cytokines and in the absence of rapamycin led to the increase in the intracellular expression of IL-17 (p-value = 0.01), while intracellular expression of IL-17 remained low following the addition of 100 nM rapamycin to the culture medium (pvalue = 0.073). The results of the functional evaluation of expanded Tregs showed relatively differences in both patient and control groups. Thus, expanded Tregs inhibited the proliferation of responder T cells in a dose-dependent manner in the control group (p-value = 0.028), while in the patient group this inhibitory effect was not significant (p-value = 0.055). CONCLUSION: Tregs isolated from beta-TM patients have poorer inhibitory performance than Tregs isolated from healthy individuals. Also, we concluded that rapamycin stabilizes the Treg population by inhibiting the production of IL-17, all necessitating the administration of appropriate immunosuppressive drugs in patients receiving Treg therapy.