The anti-SARS-CoV-2 BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation and expression of embryo-fetal globin genes in human erythroleukemia K562 cells.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causative of the ongoing coronavirus disease 2019 (COVID-19) pandemic. The SARS-CoV-2 Spike protein (S-protein) plays an important role in the early phase of SARS-CoV-2 infection through efficient interaction with ACE2. The S-protein is produced by RNA-based COVID-19 vaccines, that were fundamental for the reduction of the viral spread within the population and the clinical severity of COVID-19. However, the S-protein has been hypothesized to be responsible for damaging cells of several tissues and for some important side effects of RNA-based COVID-19 vaccines. Considering the impact of COVID-19 and SARS-CoV-2 infection on the hematopoietic system, the aim of this study was to verify the effect of the BNT162b2 vaccine on erythroid differentiation of the human K562 cell line, that has been in the past intensively studied as a model system mimicking some steps of erythropoiesis. In this context, we focused on hemoglobin production and induced expression of embryo-fetal globin genes, that are among the most important features of K562 erythroid differentiation. We found that the BNT162b2 vaccine suppresses mithramycin-induced erythroid differentiation of K562 cells. Reverse-transcription-qPCR and Western blotting assays demonstrated that suppression of erythroid differentiation was associated with sharp inhibition of the expression of α-globin and γ-globin mRNA accumulation. Inhibition of accumulation of ζ-globin and ε-globin mRNAs was also observed. In addition, we provide in silico studies suggesting a direct interaction between SARS-CoV-2 Spike protein and Hb Portland, that is the major hemoglobin produced by K562 cells. This study thus provides information suggesting the need of great attention on possible alteration of hematopoietic parameters following SARS-CoV-2 infection and/or COVID-19 vaccination.

Effects of Sirolimus treatment on patients with ß-Thalassemia: Lymphocyte immunophenotype and biological activity of memory CD4+ and CD8+ T cells.

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. ß-Thalassemia). The objective of the present study was to analyse the activity of CD4+ and CD8+ T cells in ß-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 ß-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 ß-Thalassemia patients are considered prone to immune deficiency.

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.

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.

New Synthetic Isoxazole Derivatives Acting as Potent Inducers of Fetal Hemoglobin in Erythroid Precursor Cells Isolated from ß-Thalassemic Patients.

Induction of fetal hemoglobin (HbF) is highly beneficial for patients carrying ß-thalassemia, and novel HbF inducers are highly needed. Here, we describe a new class of promising HbF inducers characterized by an isoxazole chemical skeleton and obtained through modification of two natural molecules, geldanamycin and radicicol. After preliminary biological assays based on benzidine staining and RT-qPCR conducted on human erythroleukemic K562 cells, we employed erythroid precursors cells (ErPCs) isolated from ß-thalassemic patients. ErPCs weretreated with appropriate concentrations of isoxazole derivatives. The accumulation of globin mRNAs was studied by RT-qPCR, and hemoglobin production by HPLC. We demonstrated the high efficacy of isozaxoles in inducing HbF. Most of these derivatives displayed an activity similar to that observed using known HbF inducers, such as hydroxyurea (HU) or rapamycin; some of the analyzed compounds were able to induce HbF with more efficiency than HU. All the compounds were active in reducing the excess of free α-globin in treated ErPCs. All the compounds displayed a lack of genotoxicity. These novel isoxazoles deserve further pre-clinical study aimed at verifying whether they are suitable for the development of therapeutic protocols for ß-thalassemia.

New TMA (4,6,4'-Trimethyl angelicin) Analogues as Anti-Inflammatory Agents in the Treatment of Cystic Fibrosis Lung Disease.

A series of new-generation TMA (4,6,4'-trimethyl angelicin) analogues was projected and synthetized in order to ameliorate anti-inflammatory activity, with reduced or absent toxicity. Since the NF-κB transcription factor (TF) plays a critical role in the expression of IL-8 (Interluekin 8), a typical marker of lung inflammation in Cystic Fibrosis (CF), the use of agents able to interfere with the NF-κB pathway represents an interesting therapeutic strategy. Through preliminary EMSA experiments, we identified several new TMA derivatives able to inhibit the NF-κB/DNA complex. The selected active molecules were then analyzed to evaluate the anti-inflammatory effect using both Pseudomonas aeruginosa (PAO1) infection and TNF-alpha stimulus on the CF IB3-1 cell line. It was demonstrated that mainly two TMA analogues, GY971a mesylate salt (6-p-minophenyl-4,4'-dimethyl-angelicin) and GY964 (4-phenyl-6,4'-dimethyl-angelicin), were able to decrease the IL-8 gene expression. At the same time, these molecules were found to have no pro-apoptotic, mutagenic and phototoxic effects, facilitating our decision to test the efficacy in vivo by using a mouse model of acute P. aeruginosa lung infection. The anti-inflammatory effect of GY971a was confirmed in vivo; this derivative was able to deeply decrease the total number of inflammatory cells, the neutrophil count and the cytokine/chemokine profile in the P. aeruginosa acute infection model, without evident toxicity. Considering all the obtained and reported in vitro and in vivo pre-clinical results, GY971a seems to have interesting anti-inflammatory effects, modulating the NF-κB pathway, as well as the starting lead compound TMA, but without side effects.

Treatment of Erythroid Precursor Cells from ß-Thalassemia Patients with Cinchona Alkaloids: Induction of Fetal Hemoglobin Production.

ß-thalassemias are among the most common inherited hemoglobinopathies worldwide and are the result of autosomal mutations in the gene encoding ß-globin, causing an absence or low-level production of adult hemoglobin (HbA). Induction of fetal hemoglobin (HbF) is considered to be of key importance for the development of therapeutic protocols for ß-thalassemia and novel HbF inducers need to be proposed for pre-clinical development. The main purpose on this study was to analyze Cinchona alkaloids (cinchonidine, quinidine and cinchonine) as natural HbF-inducing agents in human erythroid cells. The analytical methods employed were Reverse Transcription quantitative real-time PCR (RT-qPCR) (for quantification of γ-globin mRNA) and High Performance Liquid Chromatography (HPLC) (for analysis of the hemoglobin pattern). After an initial analysis using the K562 cell line as an experimental model system, showing induction of hemoglobin and γ-globin mRNA, we verified whether the two more active compounds, cinchonidine and quinidine, were able to induce HbF in erythroid progenitor cells isolated from ß-thalassemia patients. The data obtained demonstrate that cinchonidine and quinidine are potent inducers of γ-globin mRNA and HbF in erythroid progenitor cells isolated from nine ß-thalassemia patients. In addition, both compounds were found to synergize with the HbF inducer sirolimus for maximal production of HbF. The data obtained strongly indicate that these compounds deserve consideration in the development of pre-clinical approaches for therapeutic protocols of ß-thalassemia.

Discovery of Novel Fetal Hemoglobin Inducers through Small Chemical Library Screening.

The screening of chemical libraries based on cellular biosensors is a useful approach to identify new hits for novel therapeutic targets involved in rare genetic pathologies, such as ß-thalassemia and sickle cell disease. In particular, pharmacologically mediated stimulation of human γ-globin gene expression, and increase of fetal hemoglobin (HbF) production, have been suggested as potential therapeutic strategies for these hemoglobinopathies. In this article, we screened a small chemical library, constituted of 150 compounds, using the cellular biosensor K562.GR, carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and ß-globin gene promoters, respectively. Then the identified compounds were analyzed as HbF inducers on primary cell cultures, obtained from ß-thalassemia patients, confirming their activity as HbF inducers, and suggesting these molecules as lead compounds for further chemical and biological investigations.

Altered erythroid-related miRNA levels as a possible novel biomarker for detection of autologous blood transfusion misuse in sport.

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.

Surface plasmon resonance based analysis of the binding of LYAR protein to the rs368698783 (G>A) polymorphic Aγ-globin gene sequences mutated in ß-thalassemia.

Recent studies have identified and characterized a novel putative transcriptional repressor site in a 5' untranslated region of the Aγ-globin gene that interacts with the Ly-1 antibody reactive clone (LYAR) protein. LYAR binds the 5'-GGTTAT-3' site of the Aγ-globin gene, and this molecular interaction causes repression of gene transcription. In ß-thalassemia patients, a polymorphism has been demonstrated (the rs368698783 G>A polymorphism) within the 5'-GGTTAT-3' LYAR-binding site of the Aγ-globin gene. The major results gathered from surface plasmon resonance based biospecific interaction analysis (SPR-BIA) studies (using crude nuclear extracts, LYAR-enriched lysates, and recombinant LYAR) support the concept that the rs368698783 G>A polymorphism of the Aγ-globin gene attenuates the efficiency of LYAR binding to the LYAR-binding site. This conclusion was fully confirmed by a molecular docking analysis. This might lead to a very important difference in erythroid cells from ß-thalassemia patients in respect to basal and induced levels of production of fetal hemoglobin. The novelty of the reported SPR-BIA method is that it allows the characterization and validation of the altered binding of a key nuclear factor (LYAR) to mutated LYAR-binding sites. These results, in addition to theoretical implications, should be considered of interest in applied pharmacology studies as a basis for the screening of drugs able to inhibit LYAR-DNA interactions. This might lead to the identification of molecules facilitating induced increase of γ-globin gene expression and fetal hemoglobin production in erythroid cells, which is associated with possible reduction of the clinical severity of the ß-thalassemia phenotype. Graphical abstract.

Development and characterization of cellular biosensors for HTS of erythroid differentiation inducers targeting the transcriptional activity of γ-globin and ß-globin gene promoters.

There is a general agreement that pharmacologically mediated stimulation of human γ-globin gene expression and increase of production of fetal hemoglobin (HbF) is a potential therapeutic approach in the experimental therapy of ß-thalassemia and sickle cell anemia. Here, we report the development and characterization of cellular biosensors carrying enhanced green fluorescence protein (EGFP) and red fluorescence protein (RFP) genes under the control of the human γ-globin and ß-globin gene promoters, respectively; these dual-reporter cell lines are suitable to identify the induction ability of screened compounds on the transcription in erythroid cells of γ-globin and ß-globin genes by FACS with efficiency and reproducibility. Our experimental system allows to identify (a) HbF inducers stimulating to different extent the activity of the γ-globin gene promoter and (b) molecules that stimulate also the activity of the ß-globin gene promoter. A good correlation does exist between the results obtained by using the EGFP/RFP clones and experiments performed on erythroid precursor cells from ß-thalassemic patients, confirming that this experimental system can be employed for high-throughput screening (HTS) analysis. Finally, we have demonstrated that this dual-reporter cell line can be used for HTS in 384-well plate, in order to identify novel HbF inducers for the therapy of ß-thalassemia and sickle cell anemia. Graphical abstract.

Detection of the sickle hemoglobin allele using a surface plasmon resonance based biosensor.

Sickle Cell Disease (SCD) is a monogenic hereditary blood disorder caused by a single point mutation (ßS) in the ß globin gene resulting in an abnormal hemoglobin (HbS) that can polymerize within the erythrocytes, inducing their characteristic sickle shape. This causes hemolytic anemia and occlusive vessels for the most severe clinical status. Molecular analysis is crucial for fast and precise diagnosis of different forms of SCD, and, on the basis of underlying genotype, for supporting the most appropriate treatment options. In this context, we describe a simple and reproducible protocol for the molecular identification of the ßS mutation based on surface plasmon resonance (SPR) using the Biacore™ X100 affinity biosensor. This technology has already demonstrated its diagnostic suitability for the identification of point mutations responsible for genetic diseases such as cystic fibrosis and ß thalassemia, using a protocol based on immobilization of PCR products on the sensor chip. On the contrary, in this work we applied a SPR strategy based on an innovative interaction format, recently developed in our group also for ß thalassemia mutations. In particular, we correctly detected the ßS mutation responsible for SCD, both in homozygous and heterozygous states, after hybridization of two oligonucleotide probes (normal and mutated) for the ßS mutation, immobilized on sensor chip, with unbalanced PCR products obtained from 53 genomic DNAs carrying different ßS allele combinations.

An Aγ-globin G->A gene polymorphism associated with ß039 thalassemia globin gene and high fetal hemoglobin production.

BACKGROUND: Increase of the expression of γ-globin gene and high production of fetal hemoglobin (HbF) in ß-thalassemia patients is widely accepted as associated with a milder or even asymptomatic disease. The search for HbF-associated polymorphisms (such as the XmnI, BCL11A and MYB polymorphisms) has recently gained great attention, in order to stratify ß-thalassemia patients with respect to expectancy of the first transfusion, need for annual intake of blood, response to HbF inducers (the most studied of which is hydroxyurea). METHODS: Aγ-globin gene sequencing was performed on genomic DNA isolated from a total of 75 ß-thalassemia patients, including 31 ß039/ß039, 33 ß039/ß+IVSI-110, 9 ß+IVSI-110/ß+IVSI-110, one ß0IVSI-1/ß+IVSI-6 and one ß039/ß+IVSI-6. RESULTS: The results show that the rs368698783 polymorphism is present in ß-thalassemia patients in the 5'UTR sequence (+25) of the Aγ-globin gene, known to affect the LYAR (human homologue of mouse Ly-1 antibody reactive clone) binding site 5'-GGTTAT-3'. This Aγ(+25 G->A) polymorphism is associated with the Gγ-globin-XmnI polymorphism and both are linked with the ß039-globin gene, but not with the ß+IVSI-110-globin gene. In agreement with the expectation that this mutation alters the LYAR binding activity, we found that the Aγ(+25 G->A) and Gγ-globin-XmnI polymorphisms are associated with high HbF in erythroid precursor cells isolated from ß039/ß039 thalassemia patients. CONCLUSIONS: As a potential explanation of our findings, we hypothesize that in ß-thalassemia the Gγ-globin-XmnI/Aγ-globin-(G->A) genotype is frequently under genetic linkage with ß0-thalassemia mutations, but not with the ß+-thalassemia mutation here studied (i.e. ß+IVSI-110) and that this genetic combination has been selected within the population of ß0-thalassemia patients, due to functional association with high HbF. Here we describe the characterization of the rs368698783 (+25 G->A) polymorphism of the Aγ-globin gene associated in ß039 thalassemia patients with high HbF in erythroid precursor cells.

A validated cellular biobank for ß-thalassemia.

BACKGROUND: Cellular biobanking is a key resource for collaborative networks planning to use same cells in studies aimed at solving a variety of biological and biomedical issues. This approach is of great importance in studies on ß-thalassemia, since the recruitment of patients and collection of specimens can represent a crucial and often limiting factor in the experimental planning. METHODS: Erythroid precursor cells were obtained from 72 patients, mostly ß-thalassemic, expanded and cryopreserved. Expression of globin genes was analyzed by real time RT-qPCR. Hemoglobin production was studied by HPLC. RESULTS: In this paper we describe the production and validation of a Thal-Biobank constituted by expanded erythroid precursor cells from ß-thalassemia patients. The biobanked samples were validated for maintenance of their phenotype after (a) cell isolation from same patients during independent phlebotomies, (b) freezing step in different biobanked cryovials, (c) thawing step and analysis at different time points. Reproducibility was confirmed by shipping the frozen biobanked cells to different laboratories, where the cells were thawed, cultured and analyzed using the same standardized procedures. The biobanked cells were stratified on the basis of their baseline level of fetal hemoglobin production and exposed to fetal hemoglobin inducers. CONCLUSION: The use of biobanked cells allows stratification of the patients with respect to fetal hemoglobin production and can be used for determining the response to the fetal hemoglobin inducer hydroxyurea and to gene therapy protocols with reproducible results.

Y-chromosome identification in circulating cell-free fetal DNA using surface plasmon resonance.

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.

Inhibitory effects of SARS-CoV-2 spike protein and BNT162b2 vaccine on erythropoietin-induced globin gene expression in erythroid precursor cells from patients with ß-thalassemia.

During the recent coronavirus disease 2019 (COVID-19) pandemic several patients with ß-thalassemia have been infected by severe acute respiratory syndrome coronavirus (SARS-CoV-2), and most patients were vaccinated against SARS-CoV-2. Recent studies demonstrate an impact of SARS-CoV-2 infection on the hematopoietic system. The main objective of this study was to verify the effects of exposure of erythroid precursor cells (ErPCs) from patients with ß-thalassemia to SARS-CoV-2 spike protein (S-protein) and the BNT162b2 vaccine. Erythropoietin (EPO)-cultured ErPCs have been either untreated or treated with S-protein or BNT162b2 vaccine. The employed ErPCs were from a ß-thalassemia cellular Biobank developed before the COVID-19 pandemic. The genotypes were ß+-IVSI-110/ß+-IVSI-110 (one patient),  ß039/ß+-IVSI-110 (3 patients), and ß039/ ß039 (2 patients). After treatment with S-protein or BNT162b2 for 5 days, lysates were analyzed by high performance liquid chromatography (HPLC), for hemoglobin production, and isolated RNA was assayed by RT-qPCR, for detection of globin gene expression. The main conclusions of the results obtained are that SARS-CoV-2 S-protein and BNT162b2 vaccine (a) inhibit fetal hemoglobin (HbF) production by ß-thalassemic ErPCs and (b) inhibit γ-globin mRNA accumulation. In addition, we have performed in silico studies suggesting a high affinity of S-protein to HbF. Remarkably, the binding interaction energy of fetal hemoglobin to S-protein was comparable with that of angiotensin-converting enzyme 2 (ACE2). Our results are consistent with the hypothesis of a relevant impact of SARS-CoV-2 infection and COVID-19 vaccination on the hematopoietic system.

Increased Expression of α-Hemoglobin Stabilizing Protein (AHSP) mRNA in Erythroid Precursor Cells Isolated from ß-Thalassemia Patients Treated with Sirolimus (Rapamycin).

Background/Objectives: in ß-thalassemia, important clinical complications are caused by the presence of free α-globin chains in the erythroid cells of ß-thalassemia patients. These free α-globin chains are present in excess as a result of the lack of ß-globin chains to bind with; they tend to aggregate and precipitate, causing deleterious effects and overall cytotoxicity, maturation arrest of the erythroid cells and, ultimately, ineffective erythropoiesis. The chaperone protein α-hemoglobin-stabilizing protein (AHSP) reversibly binds with free α-globin; the resulting AHSP-αHb complex prevents aggregation and precipitation. Sirolimus (rapamycin) has been previously demonstrated to induce expression of fetal hemoglobin and decrease the excess of free α-globin chain in the erythroid cells of ß-thalassemia patients. The objective of this study was to verify whether sirolimus is also able to upregulate AHSP expression in erythroid precursor cells (ErPCs) isolated from ß-thalassemia patients. Methods: the expression of AHSP genes was analyzed by measuring the AHSP mRNA content by real-time quantitative PCR (RT-qPCR) and the AHSP protein production by Western blotting. Results: AHSP gene expression was found to be higher in ErPCs of ß-thalassemia patients in comparison to ErPCs isolated from healthy subjects. In addition, AHSP expression was further induced by treatment of ß-thalassemia ErPCs with sirolimus. Finally, AHSP mRNA was expressed at an increased level in ErPCs of sirolimus-treated ß-thalassemia patients participating in the NCT03877809 Sirthalaclin clinical trial. Conclusions: this exploratory study suggests that AHSP expression should be considered as an endpoint in clinical trials based on sirolimus.

High-Intensity Exercise Training Impact on Cardiorespiratory Fitness, Gait Ability, and Balance in Stroke Survivors: A Systematic Review and Meta-Analysis.

Stroke survivors commonly face challenges such as reduced physical activity and cardiorespiratory fitness (CRF) as well as balance and gait impairments, exacerbating their disability. While high-intensity exercise interventions have demonstrated some potential, their effects on these items remain uncertain. Therefore, our study aimed to investigate the impact of high-intensity training protocols on CRF, gait ability, and balance in stroke survivor populations. Two independent investigators systematically searched five databases for relevant RCTs following the PICO model. Through a systematic review of 25 RCTs published up to 31 May 2023, including adult first-stroke survivors, comparing high-intensity exercise training versus low-to-mild or no exercises, we evaluated outcomes such as the Six-Minute Walking Test (6 MWT), peak oxygen uptake (VO2peak), Ten-Meter Walk Test (10 MWT), Berg Balance Scale (BBS), and Timed Up and Go test (TUG). The protocol was registered in PROSPERO (registration number CRD42023456773). Meta-analyses indicated significant enhancements in CRF, as measured by 6 MWT and VO2peak, following high-intensity exercise interventions. However, no significant differences were observed in BBS, 10 MWT, and TUG. Our findings underscore the potential of high-intensity exercise interventions in ameliorating CRF among stroke survivors, although further research involving standardized protocols and long-term follow-ups is imperative to optimize rehabilitation outcomes.

Post-stroke spasticity: follow-up and functional implications of chronic long-term treatment with botulinum toxin.

BACKGROUND: Around 40% of stroke survivor develop spasticity. Plantar flexors (PF) muscles are often affected, with severe functional impairment. The treatment of choice is botulinum toxin type A (BoNT-A) combined with adjuvant treatments. The temporary pharmacological effect implies periodic reassessment and reinjection. These long-term chronic programs require monitoring the functional impact of each cycle and the clinical evolution in relation to aging and repeated interventions. AIM: Evaluating changes of functional level in patients with post-stroke spasticity treated with BoNT-A by assessing the long-term maintenance of the therapeutic efficacy. DESIGN: Retrospective longitudinal observational study. SETTING: Outpatients. POPULATION: Chronic stroke survivors undergoing BoNT-A treatment and subsequent intensive rehabilitation (10 sessions in a day-hospital regime). METHODS: Medical records of the enrolled patients were consulted. The primary endpoint was the change in PF spasticity by at least 1 point on the Modified Ashworth Scale (MAS) at each cycle. Secondary endpoints were the assessment of possible trends in gait parameters (Six Minute Walking Test [6MWT]; Timed Up and Go [TUG], and 10 Meters Walking Test [10mWT]) pre- and post-injection and at each cycle. RESULTS: Thirty-six patients were enrolled. A reduction of at least one MAS point for PF was recorded after each cycle in all subjects. A time-dependent reduction in the proportion of patients reporting an improvement higher than the minimal clinically important difference (MCID) in 6MWT and 10mWT was observed. In the case of TUG, this data kept stable at all cycles. A one-point increase in the basal functional ambulation classification (FAC) score resulted in a reduction in the probability of having a TUG improvement greater than the MCID. The opposite correlation was found for 6MWT and 10mWT. CONCLUSIONS: With the proposed treatment, the clinical significance TUG improvement remains constant throughout repeated cycles and the proportion of patients with improvement in 6MWT and 10mWT tends to decline over time. The predictive value of basal FAC on the functional variables expected improvement may provide a potential treatment targeting tool. CLINICAL REHABILITATION IMPACT: These results may deliver prognostic indication allowing an optimized integration of different post-BoNT-A rehabilitation approaches, agreeing with current evidence. Adequate monitoring and treatment protocols are crucial for the stability of functional level and may prevent excessive fluctuations.