Type I IFN drives unconventional IL-1ß secretion in lupus monocytes.

Opsonization of red blood cells that retain mitochondria (Mito+ RBCs), a feature of systemic lupus erythematosus (SLE), triggers type I interferon (IFN) production in macrophages. We report that monocytes (Mos) co-produce IFN and mature interleukin-1ß (mIL-1ß) upon Mito+ RBC opsonization. IFN expression depended on cyclic GMP-AMP synthase (cGAS) and RIG-I-like receptors' (RLRs) sensing of Mito+ RBC-derived mitochondrial DNA (mtDNA) and mtRNA, respectively. Interleukin-1ß (IL-1ß) production was initiated by the RLR antiviral signaling adaptor (MAVS) pathway recognition of Mito+ RBC-derived mtRNA. This led to the cytosolic release of Mo mtDNA, which activated the inflammasome. Importantly, mIL-1ß secretion was independent of gasdermin D (GSDMD) and pyroptosis but relied on IFN-inducible myxovirus-resistant protein 1 (MxA), which facilitated the incorporation of mIL-1ß into a trans-Golgi network (TGN)-mediated secretory pathway. RBC internalization identified a subset of blood Mo expressing IFN-stimulated genes (ISGs) that released mIL-1ß and expanded in SLE patients with active disease.

Lysis of human erythrocytes due to Piezo1-dependent cytosolic calcium overload as a mechanism of circulatory removal.

Hematopoietic stem cells surrender organelles during differentiation, leaving mature red blood cells (RBC) devoid of transcriptional machinery and mitochondria. The resultant absence of cellular repair capacity limits RBC circulatory longevity, and old cells are removed from circulation. The specific age-dependent alterations required for this apparently targeted removal of RBC, however, remain elusive. Here, we assessed the function of Piezo1, a stretch-activated transmembrane cation channel, within subpopulations of RBC isolated based on physical properties associated with aging. We subsequently investigated the potential role of Piezo1 in RBC removal, using pharmacological and mechanobiological approaches. Dense (old) RBC were separated from whole blood using differential density centrifugation. Tolerance of RBC to mechanical forces within the physiological range was assessed on single-cell and cell population levels. Expression and function of Piezo1 were investigated in separated RBC populations by monitoring accumulation of cytosolic Ca2+ and changes in cell morphology in response to pharmacological Piezo1 stimulation and in response to physical forces. Despite decreased Piezo1 activity with increasing cell age, tolerance to prolonged Piezo1 stimulation declined sharply in older RBC, precipitating lysis. Cell lysis was immediately preceded by an acute reversal of density. We propose a Piezo1-dependent mechanism by which RBC may be removed from circulation: Upon adherence of these RBC to other tissues, they are uniquely exposed to prolonged mechanical forces. The resultant sustained activation of Piezo1 leads to a net influx of Ca2+, overpowering the Ca2+-removal capacity of specifically old RBC, which leads to reversal of ion gradients, dysregulated cell hydration, and ultimately osmotic lysis.

PIEZO Ion Channels in Cardiovascular Functions and Diseases.

The cardiovascular system provides blood supply throughout the body and as such is perpetually applying mechanical forces to cells and tissues. Thus, this system is primed with mechanosensory structures that respond and adapt to changes in mechanical stimuli. Since their discovery in 2010, PIEZO ion channels have dominated the field of mechanobiology. These have been proposed as the long-sought-after mechanosensitive excitatory channels involved in touch and proprioception in mammals. However, more and more pieces of evidence point to the importance of PIEZO channels in cardiovascular activities and disease development. PIEZO channel-related cardiac functions include transducing hemodynamic forces in endothelial and vascular cells, red blood cell homeostasis, platelet aggregation, and arterial blood pressure regulation, among others. PIEZO channels contribute to pathological conditions including cardiac hypertrophy and pulmonary hypertension and congenital syndromes such as generalized lymphatic dysplasia and xerocytosis. In this review, we highlight recent advances in understanding the role of PIEZO channels in cardiovascular functions and diseases. Achievements in this quickly expanding field should open a new road for efficient control of PIEZO-related diseases in cardiovascular functions.

Assessment of stored red blood cells through lab-on-a-chip technologies for precision transfusion medicine.

Transfusion of red blood cells (RBCs) is one of the most valuable and widespread treatments in modern medicine. Lifesaving RBC transfusions are facilitated by the cold storage of RBC units in blood banks worldwide. Currently, RBC storage and subsequent transfusion practices are performed using simplistic workflows. More specifically, most blood banks follow the "first-in-first-out" principle to avoid wastage, whereas most healthcare providers prefer the "last-in-first-out" approach simply favoring chronologically younger RBCs. Neither approach addresses recent advances through -omics showing that stored RBC quality is highly variable depending on donor-, time-, and processing-specific factors. Thus, it is time to rethink our workflows in transfusion medicine taking advantage of novel technologies to perform RBC quality assessment. We imagine a future where lab-on-a-chip technologies utilize novel predictive markers of RBC quality identified by -omics and machine learning to usher in a new era of safer and precise transfusion medicine.

Real-Time Imaging Assessment of Stress-Induced Vascular Inflammation Using Heartbeat-Synchronized Motion Compensation.

BACKGROUND: Chronic mental stress accelerates atherosclerosis through complicated neuroimmune pathways, needing for advanced imaging techniques to delineate underlying cellular mechanisms. While histopathology, ex vivo imaging, and snapshots of in vivo images offer promising evidence, they lack the ability to capture real-time visualization of blood cell dynamics within pulsatile arteries in longitudinal studies. METHODS: An electrically tunable lens was implemented in intravital optical microscopy, synchronizing the focal plane with heartbeats to follow artery movements. ApoE-/- mice underwent 2 weeks of restraint stress before baseline imaging followed by 2 weeks of stress exposure in the longitudinal imaging, while nonstressed mice remained undisturbed. The progression of vascular inflammation was assessed in the carotid arteries through intravital imaging and histological analyses. RESULTS: A 4-fold reduction of motion artifact, assessed by interframe SD, and an effective temporal resolution of 25.2 Hz were achieved in beating murine carotid arteries. Longitudinal intravital imaging showed chronic stress led to a 6.09-fold (P=0.017) increase in myeloid cell infiltration compared with nonstressed mice. After 3 weeks, we observed that chronic stress intensified vascular inflammation, increasing adhered myeloid cells by 2.45-fold (P=0.031), while no significant changes were noted in nonstressed mice. Microcirculation imaging revealed increased circulating, rolling, and adhered cells in stressed mice's venules. Histological analysis of the carotid arteries confirmed the in vivo findings that stress augmented plaque area, myeloid cell and macrophage accumulation, and necrotic core volume while reducing fibrous cap thickness indicating accelerated plaque formation. We visualized the 3-dimensional structure of the carotid artery and 4-dimensional dynamics of the venules in the cremaster muscle. CONCLUSIONS: Dynamic focusing motion compensation intravital microscopy enabled subcellular resolution in vivo imaging of blood cell dynamics in beating arteries under chronic restraint stress in real time. This novel technique emphasizes the importance of advanced in vivo imaging for understanding cardiovascular disease.

Reconsidering red blood cells as the diagnostic potential for neurodegenerative disorders.

BACKGROUND: Red blood cells (RBCs) are usually considered simple cells and transporters of gases to tissues. HYPOTHESIS: However, recent research has suggested that RBCs may have diagnostic potential in major neurodegenerative disorders (NDDs). RESULTS: This review summarizes the current knowledge on changes in RBC in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and other NDDs. It discusses the deposition of neuronal proteins like amyloid-ß, tau, and α-synuclein, polyamines, changes in the proteins of RBCs like band-3, membrane transporter proteins, heat shock proteins, oxidative stress biomarkers, and altered metabolic pathways in RBCs during neurodegeneration. It also highlights the comparison of RBC diagnostic markers to other in-market diagnoses and discusses the challenges in utilizing RBCs as diagnostic tools, such as the need for standardized protocols and further validation studies. SIGNIFICANCE STATEMENT: The evidence suggests that RBCs have diagnostic potential in neurodegenerative disorders, and this study can pave the foundation for further research which may lead to the development of novel diagnostic approaches and treatments.

Novel secretory organelles of parasite origin - at the center of host-parasite interaction.

Reorganization of cell organelle-deprived host red blood cells by the apicomplexan malaria parasite Plasmodium falciparum enables their cytoadherence to endothelial cells that line the microvasculature. This increases the time red blood cells infected with mature developmental stages remain within selected organs such as the brain to avoid the spleen passage, which can lead to severe complications and cumulate in patient death. The Maurer's clefts are a novel secretory organelle of parasite origin established by the parasite in the cytoplasm of the host red blood cell in order to facilitate the establishment of cytoadherence by conducting the trafficking of immunovariant adhesins to the host cell surface. Another important function of the organelle is the sorting of other proteins the parasite traffics into its host cell. Although the organelle is of high importance for the pathology of malaria, additional putative functions, structure, and genesis remain shrouded in mystery more than a century after its discovery. In this review, we highlight our current knowledge about the Maurer's clefts and other novel secretory organelles established within the host cell cytoplasm by human-pathogenic malaria parasites and other parasites that reside within human red blood cells.

Functional asymmetry of the urea transporter UT-B in human red blood cells.

We studied urea, thiourea, and methylurea transport and interaction in human red blood cells (RBCs) under conditions of self-exchange (SE), net efflux (NE), and net influx (NI) at pH 7.2. We combined four methods, a four-centrifuge technique, the Millipore-Swinnex filtering technique, the continuous flow tube method, and a continuous pump method to measure the transport of the 14C-labeled compounds. Under SE conditions, both urea and thiourea show perfect Michaelis-Menten kinetics with half-saturation constants, K½,SE (mM), of ≈300 (urea) and ≈20 (thiourea). The solutes show no concentration-dependent saturation under NE conditions. Under NI conditions, transport displays saturation or self-inhibition kinetics with a K½,NI (mM) of ≈210 (urea) and ≈20 (thiourea). Urea, thiourea, and methylurea are competitive inhibitors of the transport of analog solutes. This study supports the hypothesis that the three compounds share the same urea transport system (UT-B). UT-B functions asymmetrically as it saturates from the outside only under SE and NI conditions, whereas it functions as a high-capacity channel-like transporter under NE conditions. When the red blood cell enters the urea-rich kidney tissue, self-inhibition reduces the urea uptake in the cell. When the cell leaves the kidney, the channel-like function of UT-B implies that intracellular urea rapidly equilibrates with external urea. The net result is that the cell during the passage in the kidney capillaries carries urea to the kidney to be excreted while the urea transfer from the kidney via the bloodstream is minimized.NEW & NOTEWORTHY The kinetics of urea transport in red blood cells was determined by means of a combination of four methods that ensures a high time resolution. In the present study, we disclose that the urea transporter UT-B functions highly asymmetric being channel-like with no saturation under conditions of net efflux and saturable under conditions of net influx and self-exchange in the concentration range 1-1,000 mM (pH 7.2 and 25-38 °C).

Altered counts and mitochondrial mass of peripheral blood leucocytes in patients with chronic hepatitis B virus infection.

Hepatitis B virus (HBV) damages liver cells through abnormal immune responses. Mitochondrial metabolism is necessary for effector functions of white blood cells (WBCs). The aim was to investigate the altered counts and mitochondrial mass (MM) of WBCs by two novel indicators of mitochondrial mass, MM and percentage of low mitochondrial membrane potential, MMPlow%, due to chronic HBV infection. The counts of lymphocytes, neutrophils and monocytes in the HBV infection group were in decline, especially for lymphocyte (p = 0.034) and monocyte counts (p = 0.003). The degraded MM (p = 0.003) and MMPlow% (p = 0.002) of lymphocytes and MM (p = 0.005) of monocytes suggested mitochondrial dysfunction of WBCs. HBV DNA within WBCs showed an extensive effect on mitochondria metabolic potential of lymphocytes, neutrophils and monocytes indicated by MM; hepatitis B e antigen was associated with instant mitochondrial energy supply indicated by MMPlow% of neutrophils; hepatitis B surface antigen, antiviral therapy by nucleos(t)ide analogues and prolonged infection were also vital factors contributing to WBC alterations. Moreover, degraded neutrophils and monocytes could be used to monitor immune responses reflecting chronic liver fibrosis and inflammatory damage. In conclusion, MM combined with cell counts of WBCs could profoundly reflect WBC alterations for monitoring chronic HBV infection. Moreover, HBV DNA within WBCs may be a vital factor in injuring mitochondria metabolic potential.

Severity of coronary artery disease is associated with diminished circANRIL expression: A possible blood based transcriptional biomarker in East Africa.

Antisense Noncoding RNA in the INK4 Locus (ANRIL) is the prime candidate gene at Chr9p21, the well-defined genetic risk locus associated with coronary artery disease (CAD). ANRIL and its transcript variants were investigated for the susceptibility to CAD in adipose tissues (AT) and peripheral blood mononuclear cells (PBMCs) of the study group and the impact of 9p21.3 locus mutations was further analysed. Expressions of ANRIL, circANRIL (hsa_circ_0008574), NR003529, EU741058 and DQ485454 were detected in epicardial AT (EAT) mediastinal AT (MAT), subcutaneous AT (SAT) and PBMCs of CAD patients undergoing coronary artery bypass grafting and non-CAD patients undergoing heart valve surgery. ANRIL expression was significantly upregulated, while the expression of circANRIL was significantly downregulated in CAD patients. Decreased circANRIL levels were significantly associated with the severity of CAD and correlated with aggressive clinical characteristics. rs10757278 and rs10811656 were significantly associated with ANRIL and circANRIL expressions in AT and PBMCs. The ROC-curve analysis suggested that circANRIL has high diagnostic accuracy (AUC: 0.9808, cut-off: 0.33, sensitivity: 1.0, specificity: 0.88). circANRIL has high diagnostic accuracy (AUC: 0.9808, cut-off: 0.33, sensitivity: 1.0, specificity: 0.88). We report the first data demonstrating the presence of ANRIL and its transcript variants expressions in the AT and PBMCs of CAD patients. circANRIL having a synergetic effect with ANRIL plays a protective role in CAD pathogenesis. Therefore, altered circANRIL expression may become a potential diagnostic transcriptional biomarker for early CAD diagnosis.

Exposure to normobaric hypoxia shapes the acute inflammatory response in human whole blood cells in vivo.

Cells of the immune defence, especially leukocytes, often have to perform their function in tissue areas that are characterized by oxygen deficiency, so-called hypoxia. Physiological hypoxia significantly affects leukocyte function and controls the innate and adaptive immune response mainly through transcriptional gene regulation via the hypoxia-inducible factors (HIFs). Multiple pathogens including components of bacteria, such as lipopolysaccharides (LPS) trigger the activation of leukocytes. HIF pathway activation enables immune cells to adapt to both hypoxic environments in physiological and inflammatory settings and modulates immune cell responses through metabolism changes and crosstalk with other immune-relevant signalling pathways. To study the mutual influence of both processes in vivo, we used a human endotoxemia model, challenging participants with an intravenous LPS injection post or prior to a 4-h stay in a hypoxic chamber with normobaric hypoxia of 10.5% oxygen. We analysed changes in gene expression in whole blood cells and determined inflammatory markers to unveil the crosstalk between both processes. Our investigations showed differentially altered gene expression patterns of HIF and target genes upon in vivo treatment with LPS and hypoxia. Further, we found evidence for effects of hypoxic priming upon inflammation in combination with immunomodulatory effects in whole blood cells in vivo. Our work elucidates the complex interplay of hypoxic and inflammatory HIF regulation in human immune cells and offers new perspectives for further clinical research.

Mouse blood cells types and aging prediction using penalized Latent Dirichlet Allocation.

BACKGROUND: Aging is a complex, heterogeneous process that has multiple causes. Knowledge on genomic, epigenomic and transcriptomic changes during the aging process shed light on understanding the aging mechanism. A recent breakthrough in biotechnology, single cell RNAseq, is revolutionizing aging study by providing gene expression profile of the entire transcriptome of individual cells. Many interesting information could be inferred from this new type of data with the help of novel computational methods. RESULTS: In this manuscript a novel statistical method, penalized Latent Dirichlet Allocation (pLDA), is applied to an aging mouse blood scRNA-seq data set. A pipeline is built for cell type and aging prediction. The sequence of models in the pipeline take scRNA-seq expression counts as input, preprocess the data using pLDA and predict the cell type and aging status. CONCLUSIONS: pLDA learns a dimension reduced representation of the expression profile. This representation allows identification of cell types and has predictability of the age of cells.

An Elevated IL10 mRNA Combined with Lower TNFA mRNA Level in Active Rheumatoid Arthritis Peripheral Blood.

We aimed to investigate the expression of pro-inflammatory cytokine genes TNFA, IL6, IL12B, IL23, IL18 and immunoregulatory genes FOXP3, TGFB1, and IL10 in the peripheral blood of patients with rheumatoid arthritis (RA) at messenger ribonucleic acid (mRNA) level. The total RNA was isolated from peripheral blood samples. Real-time quantitative PCR was used to perform TaqMan-based assays to quantify mRNAs from 8 target genes. IL23A was upregulated (1.7-fold), whereas IL6 (5-fold), FOXP3 (4-fold), and IL12B (2.56-fold) were downregulated in patients compared to controls. In addition, we found a strong positive correlation between the expression of FOXP3 and TNFA and a moderate correlation between FOXP3 and TGFB1. These data showed the imbalance of the T helper (Th) 1/Th17/ T regulatory (Treg) axis at a systemic level in RA. In cases with active disease, the IL10 gene expression was approximately 2-fold higher; in contrast, the expression of FOXP3 was significantly decreased (3.38-fold). The main part of patients with higher disease activity expressed upregulation of IL10 and downregulation of TNFA. Different disease activity cohorts could be separated based on IL10, TNFA and IL12B expression combinations. In conclusion, our results showed that active disease is associated with an elevated IL10 and lower TNFA mRNA level in peripheral blood cells of RA patients.

Red blood cells as oxygen carrier during normothermic machine perfusion of kidney grafts: Friend or foe?

Renal ex vivo normothermic machine perfusion (NMP) is under development as an assessment tool for high-risk kidney grafts and as a means of achieving more physiologically accurate organ preservation. On-going hemolysis has been reported during NMP, as this technique relies on red blood cells for oxygen delivery. In this study, we confirm the occurrence of progressive hemolysis during 6-hour kidney NMP. NMP-associated erythrostasis in the glomeruli and in peri-glomerular vascular networks points to an interaction between the red blood cells and the graft. Continuous hemolysis resulted in prooxidative changes in the perfusate, which could be quenched by addition of fresh frozen plasma. In a cell-based system, this hemolysis induced redox stress and exhibited toxic effects at high concentrations. These findings highlight the need for a more refined oxygen carrier in the context of renal NMP.

Could oxygen gradient ektacytometry help to detect sickle cell trait carriers at risk for kidney disorders or exercise-related complications?

The study of Ellsworth et al. (Br J Haematol, 2024) demonstrated the usefulness of oxygen gradient ektacytometry technique to better identify the physiological parameters that could increase the risk of sickling of red blood cells (RBCs) from sickle cell trait (SCT) carriers. Oxygen gradient ektacytometry combined with pH and osmolality modulations could help in identifying SCT carriers at risk for kidney disorders or exercise-related complications. Other factors than the percentages of haemoglobin S are probably involved in the propensity of RBCs from SCT carriers to sickle during deoxygenation. Commentary on: Ellsworth et al. Hypertonicity and/or acidosis induce marked rheological changes under hypoxic conditions in sickle trait red blood cells. Br J Haematol 2024 (Online ahead of print). doi: 10.1111/bjh.19669.

Red blood cells from patients with ST-elevation myocardial infarction and elevated C-reactive protein levels induce endothelial dysfunction.

Endothelial dysfunction is an early consequence of vascular inflammation and a driver of coronary atherosclerotic disease leading to myocardial infarction. The red blood cells (RBCs) mediate endothelial dysfunction in patients at cardiovascular risk, but their role in patients with acute myocardial infarction is unknown. This study aimed to investigate if RBCs from patients with ST-elevation myocardial infarction (STEMI) induced endothelial dysfunction and the role of systemic inflammation in this effect. RBCs from patients with STEMI and aged-matched healthy controls were co-incubated with rat aortic segments for 18h followed by evaluation of endothelium-dependent (EDR) and -independent relaxation (EIDR). RBCs and aortic segments were also analyzed for arginase and oxidative stress. The patients were divided into groups depending on C-reactive protein (CRP) levels at admission. RBCs from patients with STEMI and CRP levels >2 mg/L induced impairment of EDR, but not EIDR, compared to RBCs from STEMI and CRP <2 mg/L and healthy controls. Aortic expression of arginase 1 was increased following incubation with RBCs from patients with STEMI and CRP >2, and arginase inhibition prevented the RBC-induced endothelial dysfunction. RBCs from patients with STEMI and CRP >2 had increased reactive oxygen species compared to RBCs from patients with CRP <2 and healthy controls. Vascular inhibition of NADPH oxidases and increased dismutation of superoxide improved EDR. RBCs from patients with STEMI and low-grade inflammation induce endothelial dysfunction through a mechanism involving arginase 1 as well and increased RBC and vascular superoxide by NADPH oxidases.

Single-cell RNA sequencing and immune repertoire analysis revealed dynamic immune characteristics associated with peripheral blood during sepsis.

Sepsis is a potentially fatal condition arising from an abnormal immune response to an infection, which can result in organ failure and even death. To explore the mechanism underlying the dysregulated immune response during sepsis and identify potential therapeutic targets, single-cell RNA sequencing (scRNA-seq) and immune repertoire analysis were conducted to depict the cellular landscape of peripheral blood cells in septic mice. We observed significant alterations in the number and proportion of peripheral blood cell populations driven by sepsis. By combining single-cell gene expression profiles and B cell receptor (BCR) repertoire analysis, we discerned that infection inflicted serious damage on the antigen presentation ability of B cells and the diversity of BCR in a short time. In addition, we found that the cecal ligation and puncture procedure in mice inhibited the communication signals of CD4+ and CD8+ T cells and decreased the interactions between B cells and other cells. Our study provides detailed insights into the dynamic changes in the biological characteristics of peripheral blood cells driven by sepsis and provides important advances in our understanding of immune disorders during sepsis.

Engineering Synthetic Erythrocytes as Next-Generation Blood Substitutes.

Blood scarcity is one of the main causes of healthcare disruptions worldwide, with blood shortages occurring at an alarming rate. Over the last decades, blood substitutes has aimed at reinforcing the supply of blood, with several products (e.g., hemoglobin-based oxygen carriers, perfluorocarbons) achieving a limited degree of success. Regardless, there is still no widespread solution to this problem due to persistent challenges in product safety and scalability. In this Review, we describe different advances in the field of blood substitution, particularly in the development of artificial red blood cells, otherwise known as engineered erythrocytes. We categorize the different strategies into natural, synthetic, or hybrid approaches, and discuss their potential in terms of safety and scalability. We identify synthetic engineered erythrocytes as the most powerful approach, and describe erythrocytes from a materials engineering perspective. We review their biological structure and function, as well as explore different methods of assembling a material-based cell. Specifically, we discuss how to recreate size, shape, and deformability through particle fabrication, and how to recreate the functional machinery through synthetic biology and nanotechnology. We conclude by describing the versatile nature of synthetic erythrocytes in medicine and pharmaceuticals and propose specific directions for the field of erythrocyte engineering.

Early administration of umbilical cord blood cells following brief high tidal volume ventilation in preterm sheep: a cautionary tale.

BACKGROUND: Umbilical cord blood (UCB) cells are a promising treatment for preterm brain injury. Access to allogeneic sources of UCB cells offer the potential for early administration to optimise their therapeutic capacities. As preterm infants often require ventilatory support, which can contribute to preterm brain injury, we investigated the efficacy of early UCB cell administration following ventilation to reduce white matter inflammation and injury. METHODS: Preterm fetal sheep (0.85 gestation) were randomly allocated to no ventilation (SHAM; n = 5) or 15 min ex utero high tidal volume ventilation. One hour following ventilation, fetuses were randomly allocated to i.v. administration of saline (VENT; n = 7) or allogeneic term-derived UCB cells (24.5 ± 5.0 million cells/kg; VENT + UCB; n = 7). Twenty-four hours after ventilation, lambs were delivered for magnetic resonance imaging and post-mortem brain tissue collected. Arterial plasma was collected throughout the experiment for cytokine analyses. To further investigate the results from the in vivo study, mononuclear cells (MNCs) isolated from human UCB were subjected to in vitro cytokine-spiked culture medium (TNFα and/or IFNγ; 10 ng/mL; n = 3/group) for 16 h then supernatant and cells collected for protein and mRNA assessments respectively. RESULTS: In VENT + UCB lambs, systemic IFNγ levels increased and by 24 h, there was white matter neuroglial activation, vascular damage, reduced oligodendrocytes, and increased average, radial and mean diffusivity compared to VENT and SHAM. No evidence of white matter inflammation or injury was present in VENT lambs, except for mRNA downregulation of OCLN and CLDN1 compared to SHAM. In vitro, MNCs subjected to TNFα and/or IFNγ displayed both pro- and anti-inflammatory characteristics indicated by changes in cytokine (IL-18 & IL-10) and growth factor (BDNF & VEGF) gene and protein expression compared to controls. CONCLUSIONS: UCB cells administered early after brief high tidal volume ventilation in preterm fetal sheep causes white matter injury, and the mechanisms underlying these changes are likely dysregulated responses of the UCB cells to the degree of injury/inflammation already present. If immunomodulatory therapies such as UCB cells are to become a therapeutic strategy for preterm brain injury, especially after ventilation, our study suggests that the inflammatory state of the preterm infant should be considered when timing UCB cells administration.

Circular RNAs exhibit limited evidence for translation, or translation regulation of the mRNA counterpart in terminal hematopoiesis.

Each day, about 1012 erythrocytes and platelets are released into the bloodstream. This substantial output from hematopoietic stem cells is tightly regulated by transcriptional and epigenetic factors. Whether and how circular RNAs (circRNAs) contribute to the differentiation and/or identity of hematopoietic cells is to date not known. We recently reported that erythrocytes and platelets contain the highest levels and numbers of circRNAs among hematopoietic cells. Here, we provide the first detailed analysis of circRNA expression during erythroid and megakaryoid differentiation. CircRNA expression not only significantly increased upon enucleation, but also had limited overlap between progenitor cells and mature cells, suggesting that circRNA expression stems from regulated processes rather than resulting from mere accumulation. To study circRNA function in hematopoiesis, we first compared the expression levels of circRNAs with the translation efficiency of their mRNA counterpart. We found that only one out of 2531 (0.04%) circRNAs associated with mRNA-translation regulation. Furthermore, irrespective of thousands of identified putative open reading frames, deep ribosome-footprinting sequencing, and mass spectrometry analysis provided little evidence for translation of endogenously expressed circRNAs. In conclusion, circRNAs alter their expression profile during terminal hematopoietic differentiation, yet their contribution to regulate cellular processes remains enigmatic.