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Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β‐Thalassemia). The objective of the present study was to analyse the activity of CD4+ and CD8+ T cells in β‐Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN‐γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4+ and CD8+ T cells, respectively. The main results of the present study are that treatment of β‐Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4+ and CD8+ T cells releasing IFN‐γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β‐Thalassemia patients are considered prone to immune deficiency. [ABSTRACT FROM AUTHOR]

Background: Autologous blood transfusion (ABT) is a performance-enhancing method prohibited in sport; its detection is a key issue in the field of anti-doping. Among novel markers enabling ABT detection, microRNAs (miRNAs) might be considered a promising analytical tool.Study Design and Methods: We studied the changes of erythroid-related microRNAs following ABT, to identify novel biomarkers. Fifteen healthy trained males were studied from a population of 24 subjects, enrolled and randomized into a Transfusion (T) and a Control (C) group. Seriated blood samples were obtained in the T group before and after the two ABT procedures (withdrawal, with blood refrigerated or cryopreserved, and reinfusion), and in the C group at the same time points. Traditional hematological parameters were assessed. Samples were tested by microarray analysis of a pre-identified set of erythroid-related miRNAs.Results: Hematological parameters showed moderate changes only in the T group, particularly following blood withdrawal. Among erythroid-related miRNAs tested, following ABT a pool of 7 miRNAs associated with fetal hemoglobin and regulating transcriptional repressors of gamma-globin gene was found stable in C and differently expressed in three out of six T subjects in the completed phase of ABT, independently from blood conservation. Particularly, two or more erythropoiesis-related miRNAs within the shortlist constituted of miR-126-3p, miR-144-3p, miR-191-3p, miR-197-3p, miR-486-3p, miR-486-5p, and miR-92a-3p were significantly upregulated in T subjects after reinfusion, with a person-to-person variability but with congruent changes.Conclusions: This study describes a signature of potential interest for ABT detection in sports, based on the analysis of miRNAs associated with erythroid features. [ABSTRACT FROM AUTHOR]

Objective: Since the discovery of cell-free fetal DNA (cffDNA) in maternal plasma, diagnostic non-invasive prenatal methods have been developed or optimized for fetal sex determination and identification of genetic diseases. As far as fetal sex determination, this might be important for therapeutic intervention on sex-associated pathologies such as Duchenne muscular dystrophy, hemophilia and congenital adrenal hyperplasia. Surface plasmon resonance (SPR)-based biosensors might be useful for these studies, because they allow to monitor the molecular interactions in real-time providing qualitative and quantitative information, through kinetics, affinity and concentration analyses.Methods: The Biacore™ X100 has been applied to identify Y-chromosome sequence in cffDNA obtained from plasma samples of 26 pregnant women at different gestational ages. We have performed SPR-based analysis of SRY PCR products using SRY-specific probes immobilized on the sensor chip.Results: We have demonstrated that there is a statistically significant difference between samples collected by pregnancies carrying male or female fetuses. Moreover, cffDNA obtained at early gestational ages and not detectable by conventional quantitative real-time PCR can be discriminated with high accuracy and reliability using SPR-based biosensors.Conclusions: These data, in addition to their direct applicability in more extensive diagnostic trials, should be considered as the basis of future developments. [ABSTRACT FROM AUTHOR]

Alternative splicing of the locus AβH-J-J generates functionally distinct proteins: the enzyme aspartyl (asparaginyl) β-hydroxylase, humbug and junctate (truncated homologs of aspartyl (asparaginyl) β-hydroxylase with a role in calcium regulation), and junctin (a structural protein of the sarcoplasmic reticulum membrane). Aspartyl (asparaginyl) β-hydroxylase and humbug are overexpressed in a broad range of malignant neoplasms. We have previously reported the gene structure of this locus, showing the presence of two putative promoters, P1 and P2, and characterized the P2 sequences, directing tissue-specific transcription of junctin, aspartyl (asparaginyl) β-hydroxylase and junctate. In addition, aspartyl (asparaginyl) β-hydroxylase and humbug are expressed from exon 1 by the P1 promoter. The present study identifies and functionally characterizes the P1 promoter activity of the AβH-J-J locus. We demonstrate that mRNAs from the P1 promoter are actively transcribed in all the human tissues and cell lines analyzed, and define the transcription start point in HeLa and RD cells. To investigate the transcription mechanism we cloned 1.7 kb upstream of exon 1 from a human BAC clone, and produced progressively deleted reporter constructs. Our results showed that: (a) the 1.7 kb fragment was a powerful activator of the reporter gene in human hepatoblastoma (HepG2) and human embryonic rhabdomyosarcoma (RD) cell lines; (b) 512 bp upstream of the transcription start site were essential for maximal promoter activity; and (c) progressive deletions from − 512 resulted in gradually decreased reporter expression. The region responsible for maximal transcription contains at least 12 GC boxes homologous to binding sequences of specific transcription factor 1 (Sp1); by electrophoretic mobility shift assay and supershift analysis, we identified three GC-rich elements that bind Sp transcription factor family nuclear factors with very high efficiency. A functional role of Sp transcription factors in upregulating P1-directed transcription was demonstrated by analysis of the effects of: (a) in vitro mutagenesis of the Sp1 transcription factor binding sites; (b) transfection with Sp transcription factor 1/3 expression vectors; and (c) treatment with decoy oligonucleotides targeting Sp transcription factors. In addition, Sp1 and Sp3 transcription factor chromatin immunoprecipitation demonstrated in vivo binding of these proteins to P1 promoter. Our results suggest that Sp transcription factors positively regulate the core of the P1 promoter, and the comparison of the two promoters of the AβH-J-J locus demonstrates that they are very different with regard to transcriptional efficiency and ability to direct tissue-specific transcription. [ABSTRACT FROM AUTHOR]

The human AβH-J-J locus is a genomic sequence which generates three functionally distinct proteins, the enzyme aspartyl-β-hydroxylase (AβH), the structural protein of sarcoplasmic reticulum junctin, and the membrane-bound calcium binding protein junctate. The first and second exons are mutually exclusive when mature mRNAs are produced. Moreover, the use of different splice donors has been shown to be involved in the generation of protein diversity by alternative splicing. As to transcriptional regulation, two promoters (P1 and P2) were identified. When the P1 and P2 promoter sequences are compared, important differences are clearly detectable. The most interesting result emerging from studies focused on the P2 promoter is that the calcium-dependent transcriptional factor MEF-2 activates the transcription of junctin, junctate, and AβH in excitable tissues and, to a lesser extent, in kidney. No Sp1 binding sites are present in the P2 promoter. In contrast, P1 promoter contains GC-rich sequences, which have homologies with the Sp1 consensus binding site. [ABSTRACT FROM AUTHOR]

We studied the effects of rapamycin on cultures of erythroid progenitors derived from the peripheral blood of 10 β-thalassaemia patients differing widely with respect to their potential to produce foetal haemoglobin (HbF). For this, we employed the two-phase liquid culture procedure for growing erythroid progenitors, high performance liquid chromatography for analysis of HbF production and reverse transcription polymerase chain reaction for quantification of the accumulation of globin mRNAs. The results demonstrated that rapamycin induced an increase of HbF in cultures from all the β-thalassaemia patients studied and an increase of their overall Hb content/cell. The inducing effect of rapamycin was restricted to γ-globin mRNA accumulation, being only minor for β-globin and none for α-globin mRNAs. The ability of rapamycin to preferentially increase γ-globin mRNA content and production of HbF in erythroid precursor cells from β-thalassaemia patients is of great importance as this agent (also known as sirolimus or rapamune) is already in clinical use as an anti-rejection agent following kidney transplantation. These data suggest that rapamycin warrants further evaluation as a potential therapeutic drug in β-thalassaemia and sickle cell anaemia. [ABSTRACT FROM AUTHOR]

Inhibitors of the mammalian target of rapamycin (mTOR) have been proposed to improve vaccine responses, especially in the elderly. Accordingly, testing mTOR inhibitors (such as Sirolimus) and other geroprotective drugs might be considered a key strategy to improve overall health resilience of aged populations. In this respect, Sirolimus (also known as rapamycin) is of great interest, in consideration of the fact that it is extensively used in routine therapy and in clinical studies for the treatment of several diseases. Recently, Sirolimus has been considered in laboratory and clinical studies aimed to find novel protocols for the therapy of hemoglobinopathies (e.g. β-Thalassemia). The objective of the present study was to analyse the activity of CD4 + and CD8 + T cells in β-Thalassemia patients treated with Sirolimus, taking advantages from the availability of cellular samples of the NCT03877809 clinical trial. The approach was to verify IFN-γ releases following stimulation of peripheral blood mononuclear cells (PBMCs) to stimulatory CEF and CEFTA peptide pools, stimulatory for CD4 + and CD8 + T cells, respectively. The main results of the present study are that treatment of β-Thalassemia patients with Sirolimus has a positive impact on the biological activity and number of memory CD4 + and CD8 + T cells releasing IFN-γ following stimulation with antigenic stimuli present in immunological memory. These data are to our knowledge novel and in our opinion of interest, in consideration of the fact that β-Thalassemia patients are considered prone to immune deficiency., (© 2022 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

In several types of thalassemia (including beta(0)39-thalassemia), stop codon mutations lead to premature translation termination and to mRNA destabilization through nonsense-mediated decay. Drugs (for instance aminoglycosides) can be designed to suppress premature termination, inducing a ribosomal readthrough. These findings have introduced new hopes for the development of a pharmacologic approach to the cure of this disease. However, the effects of aminoglycosides on globin mRNA carrying beta-thalassemia stop mutations have not yet been investigated. In this study, we have used a lentiviral construct containing the beta(0)39-thalassemia globin gene under control of the beta-globin promoter and a LCR cassette. We demonstrated by fluorescence-activated cell sorting (FACS) analysis the production of beta-globin by K562 cell clones expressing the beta(0)39-thalassemia globin gene and treated with G418. More importantly, after FACS and high-performance liquid chromatography (HPLC) analyses, erythroid precursor cells from beta(0)39-thalassemia patients were demonstrated to be able to produce beta-globin and adult hemoglobin after treatment with G418. This study strongly suggests that ribosomal readthrough should be considered a strategy for developing experimental strategies for the treatment of beta(0)-thalassemia caused by stop codon mutations. Am. J. Hematol., 2009. (c) 2009 Wiley-Liss, Inc.

Nonsense mutations, giving rise to UAA, UGA and UAG stop codons within the coding region of mRNAs, promote premature translational termination and are the leading cause of approx. 30% of inherited diseases, including cystic fibrosis, Duchenne muscular dystrophy and thalassaemia. For instance, in beta(0)39-thalassaemia the CAG (glutamine) codon is mutated to the UAG stop codon, leading to premature translation termination and to mRNA destabilization through the well-described NMD (nonsense-mediated mRNA decay). In order to develop an approach facilitating translation and, therefore, protection from NMD, aminoglycoside antibiotics have been tested on mRNAs carrying premature stop codons. These drugs decrease the accuracy in the codon-anticodon base-pairing, inducing a ribosomal read-through of the premature termination codons. Interestingly, recent papers have described drugs designed and produced for suppressing premature translational termination, inducing a ribosomal read-through of premature but not normal termination codons. These findings have introduced new hopes for the development of a pharmacological approach to the therapy of beta(0)39-thalassaemia. In this context, we started the development of a cellular model of the beta(0)39-thalassaemia mutation that could be used for the screening of a high number of aminoglycosides and analogous molecules. To this aim, we produced a lentiviral construct containing the beta(0)39-thalassaemia globin gene under a minimal LCR (locus control region) control and used this construct for the transduction of K562 cells, subsequently subcloned, with the purpose to obtain several K562 clones with different integration copies of the construct. These clones were then treated with Geneticin (also known as G418) and other aminoglycosides and the production of beta-globin was analysed by FACS analysis. The results obtained suggest that this experimental system is suitable for the characterization of correction of the beta(0)39-globin mutation causing beta-thalassaemia.