Towards manufactured red blood cells for the treatment of inherited anemia

Patients with inherited anemia and hemoglobinopathies (such as sickle cell disease and β-thalassemia) are treated with red blood cell (RBC) transfusions to alleviate their symptoms. Some of these patients may have rare blood group types or go on to develop alloimmune reactions, which can make it difficult to source compatible blood in the donor population. Laboratory-grown RBC represent a particularly attractive alternative which could satisfy an unmet clinical need. The challenge, however, is to produce - from a limited number of stem cells - the 2x1012 RBC required for a standard adult therapeutic dose. Encouraging progress has been made in RBC production from adult stem cells under good manufacturing practice. In 2011, the Douay group conducted a successful proof-of-principle mini-transfusion of autologous manufactured RBC in a single volunteer. In the UK, a trial is planned to assess whether manufactured RBC are equivalent to RBC produced naturally in donors, by testing an allogeneic mini-dose of laboratory-grown manufactured RBC in multiple volunteers. This review discusses recent progress in the erythroid culture field as well as opportunities for further scaling up of manufactured RBC production for transfusion practice

Resuming Training in High-Level Athletes After Mild COVID-19 Infection: A Multicenter Prospective Study (ASCCOVID-19)

BACKGROUND: There is a paucity of data on cardiovascular sequelae of asymptomatic/mildly symptomatic SARS-Cov-2 infections (COVID). OBJECTIVES: The aim of this prospective study was to characterize the cardiovascular sequelae of asymptomatic/mildly symptomatic COVID-19 among high/elite-level athletes. METHODS: 950 athletes (779 professional French National Rugby League (F-NRL) players; 171 student athletes) were included. SARS-Cov-2 testing was performed at inclusion, and F-NRL athletes were intensely followed-up for incident COVID-19. Athletes underwent ECG and biomarker profiling (D-Dimer, troponin, C-reactive protein). COVID(+) athletes underwent additional exercise testing, echocardiography and cardiac magnetic resonance imaging (CMR). RESULTS: 285/950 athletes (30.0%) had mild/asymptomatic COVID-19 [79 (8.3%) at inclusion (COVID(+)(prevalent)); 206 (28.3%) during follow-up (COVID(+)(incident))]. 2.6% COVID(+) athletes had abnormal ECGs, while 0.4% had an abnormal echocardiogram. During stress testing (following 7-day rest), COVID(+) athletes had a functional capacity of 12.8 ± 2.7 METS with only stress-induced premature ventricular ectopy in 10 (4.3%). Prevalence of CMR scar was comparable between COVID(+) athletes and controls [COVID(+) vs. COVID(-); 1/102 (1.0%) vs 1/28 (3.6%)]. During 289 ± 56 days follow-up, one athlete had ventricular tachycardia, with no obvious link with a SARS-CoV-2 infection. The proportion with troponin I and CRP values above the upper-limit threshold was comparable between pre- and post-infection (5.9% vs 5.9%, and 5.6% vs 8.7%, respectively). The proportion with D-Dimer values above the upper-limit threshold increased when comparing pre- and post-infection (7.9% vs 17.3%, P = 0.01). CONCLUSION: The absence of cardiac sequelae in pauci/asymptomatic COVID(+) athletes is reassuring and argues against the need for systematic cardiac assessment prior to resumption of training (clinicaltrials.gov; NCT04936503).L'Institut de Rythmologie et modélisation Cardiaqu

The majority of the in vitro erythroid expansion potential resides in CD34− cells, outweighing the contribution of CD34+ cells and significantly increasing the erythroblast yield from peripheral blood samples

The study of human erythropoiesis in health and disease requires a robust culture system that consistently and reliably generates large numbers of immature erythroblasts that can be induced to differentiate synchronously. We describe a culture method modified from Leberbauer et al. (2005) and obtain a homogenous population of erythroblasts from peripheral blood mononuclear cells (PBMC) without prior purification of CD34+ cells. This pure population of immature erythroblasts can be expanded to obtain 4×108 erythroblasts from 1×108 PBMC after 13–14 days in culture. Upon synchronized differentiation, high levels of enucleation (80–90%) and low levels of cell death (<10%) are achieved. We compared the yield of erythroblasts obtained from PBMC, CD34+ cells or PBMC depleted of CD34+ cells and show that CD34− cells represent the most significant early erythroid progenitor population. This culture system may be particularly useful for investigating the pathophysiology of anemic patients where only small blood volumes are available

BNIP3L/NIX regulates both mitophagy and pexophagy

19 p.-6 fig.Mitochondria and peroxisomes are closely related metabolic organelles, both in terms of origin and in terms of function. Mitochondria and peroxisomes can also be turned over by autophagy, in processes termed mitophagy and pexophagy, respectively. However, despite their close relationship, it is not known if both organelles are turned over under similar conditions, and if so, how this might be coordinated molecularly. Here, we find that multiple selective autophagy pathways are activated upon iron chelation and show that mitophagy and pexophagy occur in a BNIP3L/NIX-dependent manner. We reveal that the outer mitochondrial membrane-anchored NIX protein, previously described as a mitophagy receptor, also independently localises to peroxisomes and drives pexophagy. We show this process happens in vivo, with mouse tissue that lacks NIX having a higher peroxisomal content. We further show that pexophagy is stimulated under the same physiological conditions that activate mitophagy, including cardiomyocyte and erythrocyte differentiation. Taken together, our work uncovers a dual role for NIX, not only in mitophagy but also in pexophagy, thus illustrating the interconnection between selective autophagy pathways.This work was funded by an EMBO Long-Term Fellowship (LPW; ALTF 1077-2018), a grant from the Medical Research Council, UK (IGG; MC_UU_00018/2) and by Ministerio de Ciencia, Innovación y Universidades (MCIU), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) PGC2018-098557-B-I00 to PB.Peer reviewe

Identification of Genes Up-Regulated in Urothelial Tumors: The 67-kd Laminin Receptor and Tumor-Associated Trypsin Inhibitor

Studies investigating changes in gene expression in urothelial carcinoma have generally compared tumors of different stages and grades but comparisons between low-grade, noninvasive tumors and normal urothelium are needed to identify genes involved in early tumor development. We isolated the urothelium from a low-grade tumor and corresponding normal mucosa by laser capture microdissection on frozen sections. The RNA extracted was amplified to generate suppressive subtractive cDNA libraries. Random sequencing of cDNA clones identified ∼100 unique species. Of these 83% were known genes, 15% had homology to genes with an unknown function in humans, and 2% did not show homology to any published gene sequence. Two of the known genes, the 67-kd laminin receptor (67LR) and tumor-associated trypsin inhibitor (TATI), had previously been associated with metastatic progression in many tumor types, although 67LR has not been investigated in urothelial tumors. Immunolabeling of the original tissue with antibodies against these two genes confirmed overexpression, validating our strategy: 67LR was not expressed in the normal urothelium but was present in the tumor, whereas TATI expression was confined to umbrella cells in the normal urothelium, but extended to all cell layers in the tumor. We investigated both markers further in a separate series of tumors of different stages and grades. TATI was more consistently overexpressed than 67LR in all tumor grades and stages. Levels of secreted TATI were significantly higher in urine samples from patients with tumors compared to controls. Our strategy, combining laser capture microdissection and cDNA library construction, has identified genes that may be involved in the early phases of urothelial tumor development rather than with disease progression, highlighting the importance of comparing tumor with normal rather than just tumors of different stages and grades

Characteristic phenotypes associated with Congenital Dyserythropoietic Anemia (Type II) manifest at different stages of erythropoiesis

Congenital dyserythropoietic anemia type II is an autosomally recessive form of hereditary anemia caused by SEC23B gene mutations. Patients exhibit characteristic phenotypes including multinucleate erythroblasts, erythrocytes with hypoglycosylated membrane proteins and an apparent double plasma membrane. Despite ubiquitous expression of SEC23B, the effects of mutations in this gene are confined to the erythroid lineage and the basis of this erythroid specificity remains to be defined. In addition, little is known regarding the stage at which the disparate phenotypes of this disease manifest during erythropoiesis. We employ an in vitro culture system to monitor the appearance of the defining phenotypes associated with congenital dyserythropoietic anemia type II during terminal differentiation of erythroblasts derived from small volumes of patient peripheral blood. Membrane protein hypoglycosylation was detected by the basophilic stage, preceding the onset of multinuclearity in orthochromatic erythroblasts that occurs coincident with the loss of secretory pathway proteins including SEC23A during erythropoiesis. Endoplasmic reticulum remnants were observed in nascent reticulocytes of both diseased and healthy donor cultures but were lost upon further maturation of normal reticulocytes, implicating a defect of ER clearance during reticulocyte maturation in congenital dyserythropoietic anemia type II. We also demonstrate distinct isoform and species-specific expression profiles of SEC23 during terminal erythroid differentiation and identify a prolonged expression of SEC23A in murine erythropoiesis compared to humans. We propose that SEC23A is able to compensate for the absence of SEC23B in mouse erythroblasts, providing a basis for the absence of phenotype within the erythroid lineage of a recently described SEC23B knockout mouse