Human red blood cells express the RNA sensor TLR7 and bind viral RNA

Red blood cells (RBCs) express the nucleic acid-sensing toll-like receptor 9 (TLR9) and bind CpG-containing DNA. However, whether human RBCs express other nucleic acid-sensing TLRs and bind RNA is unknown. Here we show that human RBCs express the RNA sensor, TLR7. TLR7 is present on the red cell membrane and associates with the RBC membrane protein Band 3. RBCs bind synthetic single-stranded RNA and RNA from pathogenic single-stranded RNA viruses. RNA acquisition by RBCs is attenuated by recombinant TLR7 and inhibitory oligonucleotides. Thus, RBCs may represent a previously unrecognized reservoir for RNA, although how RNA-binding by RBCs modulates the immune response has yet to be elucidated. These findings add to the growing list of non-gas exchanging RBC immune functions.

Altered Ca2+ Homeostasis in Red Blood Cells of Polycythemia Vera Patients Following Disturbed Organelle Sorting during Terminal Erythropoiesis

Over 95% of Polycythemia Vera (PV) patients carry the V617F mutation in the tyrosine kinase Janus kinase 2 (JAK2), resulting in uncontrolled erythroid proliferation and a high risk of thrombosis. Using mass spectrometry, we analyzed the RBC membrane proteome and showed elevated levels of multiple Ca2+ binding proteins as well as endoplasmic-reticulum-residing proteins in PV RBC membranes compared with RBC membranes from healthy individuals. In this study, we investigated the impact of JAK2V617F on (1) calcium homeostasis and RBC ion channel activity and (2) protein expression and sorting during terminal erythroid differentiation. Our data from automated patch-clamp show modified calcium homeostasis in PV RBCs and cell lines expressing JAK2V617F, with a functional impact on the activity of the Gárdos channel that could contribute to cellular dehydration. We show that JAK2V617F could play a role in organelle retention during the enucleation step of erythroid differentiation, resulting in modified whole cell proteome in reticulocytes and RBCs in PV patients. Given the central role that calcium plays in the regulation of signaling pathways, our study opens new perspectives to exploring the relationship between JAK2V617F, calcium homeostasis, and cellular abnormalities in myeloproliferative neoplasms, including cellular interactions in the bloodstream in relation to thrombotic events.

The Oxidative Status and Na +/K + -ATPase Activity in Obsessive-Compulsive Disorder: A Case Control Study

Background: Oxidative stress is involved in pathogenesis of some psychiatric disorders. To examine the role of oxidative stress in the etiopathogenesis of obsessive-compulsive disorder (OCD), we aimed to determine oxidative stress indices, including MDA levels in serum and red blood cells (RBC) membrane, total antioxidant capacity (TAC), serum glutathione (GSH) levels, serum antioxidant vitamins (A and E) and Na+/K+-ATPase activity in patients with the mentioned disorder vs. healthy controls.Method: 39 OCD patients diagnosed based on Diagnostic and Statistical Manual of Mental Disorders (DSM-V), and 39 volunteers’ healthy subjects were included in this study. MDA levels in serum and RBC membrane were measured using fluorimetric method. Serum TAC level, serum GSH level and Na+/K+-ATPase activity were also measured using spectrophotometric methods. Serum levels of vitamins were calculated by Reversed-phase high-performance liquid chromatography (RP-HPLC).Result: There was a significantly higher MDA level in serum (p<0.0001) and RBC membrane (p=0.002) of OCD patients compared with those in controls. A significant reduction in vitamin A (p=0.024) and vitamin E (p=0.001) levels was found in OCD patients vs. controls. There was significantly lower activity of erythrocyte membrane Na+-K+ ATPase in RBC membrane of OCD patients vs. controls (p<0.0001).Conclusion: Our findings indicate significantly higher levels MDA in both serum and RBC membrane, lower levels of serum vitamin A and E, and lower activity of membrane Na+-K+ ATPase in OCD patients compared to controls. These suggest an imbalance between oxidant and antioxidant factors in OCD patients that might play a fundamental role in the etiopathogenesis of OCD.

The protein organization of a red blood cell

SUMMARYRed blood cells (RBCs, erythrocytes) are the simplest primary human cells, lacking nuclei and major organelles, and instead employing about a thousand proteins to dynamically control cellular function and morphology in response to physiological cues. In this study, we defined a canonical RBC proteome and interactome using quantitative mass spectrometry and machine learning. Our data reveal an RBC interactome dominated by protein homeostasis, redox biology, cytoskeletal dynamics, and carbon metabolism. We validated protein complexes through electron microscopy and chemical crosslinking, and with these data, built 3D structural models of the ankyrin/Band 3/Band 4.2 complex that bridges the spectrin cytoskeleton to the RBC membrane. The model suggests spring-link compression of ankyrin may contribute to the characteristic RBC cell shape and flexibility. Taken together, our study provides an in-depth view of the global protein organization of human RBCs and serves as a comprehensive resource for future research.

Diagnosis and clinical management of red cell membrane disorders

Heterogeneous red blood cell (RBC) membrane disorders and hydration defects often present with the common clinical findings of hemolytic anemia, but they may require substantially different management, based on their pathophysiology. An accurate and timely diagnosis is essential to avoid inappropriate interventions and prevent complications. Advances in genetic testing availability within the last decade, combined with extensive foundational knowledge on RBC membrane structure and function, now facilitate the correct diagnosis in patients with a variety of hereditary hemolytic anemias (HHAs). Studies in patient cohorts with well-defined genetic diagnoses have revealed complications such as iron overload in hereditary xerocytosis, which is amenable to monitoring, prevention, and treatment, and demonstrated that splenectomy is not always an effective or safe treatment for any patient with HHA. However, a multitude of variants of unknown clinical significance have been discovered by genetic evaluation, requiring interpretation by thorough phenotypic assessment in clinical and/or research laboratories. Here we discuss genotype-phenotype correlations and corresponding clinical management in patients with RBC membranopathies and propose an algorithm for the laboratory workup of patients presenting with symptoms and signs of hemolytic anemia, with a clinical case that exemplifies such a workup.

A nanodrug system overexpressed circRNA_0001805 alleviates nonalcoholic fatty liver disease via miR-106a-5p/miR-320a and ABCA1/CPT1 axis

Our study aimed to explore the function of circRNA_0001805 in the pathogenesis of NAFLD and the underlying mechanism. A nanodrug system (GA-RM/GZ/PL) was constructed to overexpress circRNA_0001805 specifically in hepatocytes for the treatment of NAFLD. Fat droplet accumulation in cultured cells and mouse hepatic tissues was detected using Oil Red O or H&E staining. The relative expression of circRNAs, genes associated with lipogenesis was quantified by qRT-PCR. Interactions between circRNA_0001805 and miR-106a-5p/miR-320a, between miR-106a-5p/miR-320a and ABCA1/CPT1 were confirmed by dual-luciferase reporter assay. A novel metalorganic framework nanocarrier (GZ) was prepared from glycyrrhizic acid and zinc ions (Zn2+), and this nanocarrier was loaded with the circRNA_0001805 plasmid to construct a nanocore (GZ/PL). Then, this GZ/PL was coated with a galactose-modified RBC membrane (GA-RM) to generate GA-RM/GZ/PL. CircRNA_0001805 expression was downregulated in FFA-challenged primary hepatocytes, HFD-fed mice and NAFLD patients. Overexpressed circRNA_0001805 attenuated NAFLD development by suppressing lipid metabolism disorder and inflammation. CircRNA_0001805 targeted miR-106a-5p/miR-320a, which served as an upstream inhibitor of ABCA1/CPT1 and collaboratively regulated NAFLD progression. GA-RM/GZ/PL targeted hepatocytes, overexpressed circRNA_0001805, released glycyrrhizic acid to reduce the accumulation of lipids in the liver and played a synergistic role against NAFLD-induced lipid metabolism disorder. Graphical Abstract

Whole Blood Adhesion to VCAM-1 and P-Selectin and RBC Mechanical Fragility Can be Compromised in Long COVID-19 Patients with Sickle Cell Disease

Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause severe vascular complications associated with endothelial dysfunction and systemic inflammation. COVID19-specific IgG are detectable within a week of infection. Long COVID-19 has been described in patients continuing to exhibit symptoms after the virus is no longer detectable in the respiratory secretions, including fatigue, dyspnea, headache, and brain fog. The recent FAIR Health study reviewed a total of 1,959,982 COVID-19 patients for the prevalence of long COVID symptoms and reported that 23.2% had at least one post-COVID symptom [1]. The underlying biologic mechanisms of long COVID remain unclear, thus treatments are limited to symptomatic relief and supportive care. Many long COVID symptoms are consistent with systemic inflammation and impaired oxygen delivery observed in individuals with sickle cell disease (SCD), in turn associated with elevated blood cell adhesion and decreased red blood cell (RBC) stability. The aim of this study was to determine if deleterious changes in in blood cell properties related to adhesion and membrane stability under stress can be associated with the symptoms of long COVID-19. In this work we evaluated 7 SCD patients that were diagnosed with SARS-Cov-2 and tracked their recovery using semiquantitative IgG and blood cell function assays. Methods: Blood samples were collected by the Foundation for Sickle Cell Disease (SCD) Research from SCD (homozygous SS, n=6) patients coming for regular or urgent clinic visit with SARS-CoV-2 serological and blood cell functions tests performed per the standard of care. Semiquantitative IgG assay was performed using DXi-80 (Beckman Coulter). Flow adhesion of whole blood to VCAM-1 (FA-WB-VCAM)and P-Selectin (FA-WB-Psel) substrates were determined by counting the cells that remain adherent in a microfluidics channel after perfusion with whole blood 1:1 diluted with HBSS buffer and washed by reversed flow at 1 dyne/cm 2. Red blood cell mechanical fragility (RBC MF) was measured as hemolysis induced by an oscillating cylindrical magnet with periodic non-invasive probing of cell-free hemoglobin fraction. Six individuals with SCD recovering from SARS-Cov-2 with biomarker data available both before and for more than 3 months after the infection (179±62 days) were included in the study. Results: IgG levels varied from less than 0.1 to 37, with positive values being defined as IgG &gt; 1. The median estimated half-life of IgG decline was 53 days ranging from 25 to 90 days (the last, for the hospitalized patient). Averaged for IgG positive (IgG+) and IgG negative (IgG-) conditions, combining pre- and post-infection IgG- conditions, values of patient hemoglobin (Hb), FA-WB-VCAM, FA-WB-Psel, and RBC MF cell properties lacked statistical significance (under both a paired t-test and population statistics). Hb levels remained essentially unchanged regardless of the time from infection or IgG status. However, FA-WB-VCAM, FA-WB-Psel, and RBC MF were all significantly elevated after SARS-Cov-2 seroconversion and remained elevated despite declining IgG levels (e.g., Fig. 1). These increases in biomarker values were statistically significant for both FA-WB-VCAM and RBC MF, and were approaching significance for FA-WB-Psel (p&lt;0065). These increases were highly patient-specific with potential return to pe-infection values observed in some cases at about 5-6 months after the infection. A qualitative review of the medical records indicated a new subjective report of fatigue in 5 of 6 patients. Longer observations are required to determine if abnormal blood cell adhesive properties and RBC membrane instability are mechanisms of long-COVID-19 pathophysiology. Conclusions: Whole blood adhesion to both p-selectin and VCAM-1 as well as RBC membrane stability can be significantly impaired in convalescent SARS-Cov-2 patients suggesting an association with long COVID-19. New and emerging treatments that modify whole blood adhesive properties and RBC membrane stability should be investigated for their potential to accelerated recovery from long COVID-19. Health F. A Detailed Study of Patients with Long-Haul COVID: An Analysis of Private Healthcare Claims; White Paper. June 15, 2021 Disclosures Tarasev: Functional Fluidics: Current holder of stock options in a privately-held company. Ferranti: Functional Fluidics: Current holder of stock options in a privately-held company. Allen: Functional Fluidics: Current Employment. Gao: Functional Fluidics: Current Employment. Topping: Functional Fluidics: Current Employment. Ferranti: Functional Fluidics: Current Employment. Makinde-Odesola: Functional Fluidics: Other: conduct research for academic program. Hines: Functional Fluidics: Current holder of stock options in a privately-held company.

PIEZO1 Mutation May Determine Early Onset of Clinical Manifestation of Anemia of Myelodysplastic Syndromes

BACKGROUND: Anemia is the most frequent cytopenia in myelodysplastic syndromes (MDS), neoplastic diseases characterized by ineffective hematopoiesis and bone marrow dysplasia. MDS usually affect elderly individuals (median age in Italy 74 years), and quite rarely concern younger patients. It is of increasing interest to verify whether "young", not therapy-related MDS patients carry germline (GM) predisposition, in order to optimize therapeutic and transplant choices. The best approach is to perform Whole Exome Sequencing (WES) instead of targeted next-generation sequencing (t-NGS) panel in cases in which MDS patients have no syndromic signs or any indicative sign of predisposing alterations. AIM: We considered a cohort of 21 consecutive MDS cases with age at diagnosis &lt; 60 years and anemia, referred to our MDS Unit for a second opinion. METHODS: After bone marrow (BM) re-evaluation and morphological confirmation of dysplasia compatible with MDS, we screened the 21 MDS patients by WES on BM-DNA or peripheral blood DNA (PB-DNA). For DNA sequencing, DNA libraries were sequenced using the NextSeq 550 Illumina System. Variant calling was carried out using GATK caller. T-NGS myeloid panel was in some cases applied to confirm somatic nature of mutations. Samples of 5/21 MDS patients and their relatives were further analyzed after WES by applying high-coverage t-NGS 86-gene custom panel for hereditary anemias on PB-DNA. WES of DNA extracted from saliva (S-DNA) samples was performed to provide a GM confirmation. In order to confirm pathogenic role in red blood cell (RBC) membrane defects of identified variants, we also performed the ektacytometry analysis that evaluates the erythrocyte deformability and the hydration status by subjecting them to an increasing osmotic gradient with constant shear stress. RESULTS: Five of 21 MDS cases (23.8%) with symptomatic anemia (3 MDS-MLD, 1 MDS-SLD, 1 MDS with isolated del5q) were identified as carriers of variants suspected responsible of RBC membrane defects in both BM/PB-DNA and S-DNA samples, where no other germline myeloid predisposing alterations were demonstrated. MDS-associated somatic variants were present in 4/5 patients (KRAS, TET2, CBL, SRSF2, U2AF1, IDH2, with VAF &gt;25 %). High coverage t-NGS analysis confirmed the presence of a heterozygous rare missense variant in PIEZO1 in probands 1 and 5; a heterozygous rare missense variant in PIEZO1 and ANK1 in proband 2; a heterozygous rare missense variant in PIEZO1 and SPTA1 in proband 3; a complex mode of inheritance in proband 4 with causative variants in G6PD, KCCN4, SPTB, and ABCB6 genes. PIEZO1 or KCNN4 gene variants result in erythrocyte dehydration and determine dehydrated hereditary stomatocytosis (DHS). Ektacytometry analysis showed a significant difference in the osmolarity curve of these patients, confirming the pathogenic role of the variants identified. Among these MDS suspected cases, only proband 4 was diagnostically re-categorized because of lack of acquired recurrent somatic mutations and presence of multiple alterations in erythroid genes causing dysplasia of erythroid precursors, masquerading as MDS. None of the patients presented a history of unexplained anemia before the onset of MDS, consistent with what shown for DHS1 patients with well-compensated hemolysis. Inheritance pattern of the identified variants according the study of the families confirmed the segregation of the pathogenic variants. CONCLUSION: Onset of de novo MDS in unusually young age should always prompt investigation of predisposing conditions. We suggest here that co-existence of inherited RBC membrane defects with MDS may determine earlier clinical manifestation of the clonal disease. Hereditary anemias, in particular xerocytosis, may mimic MDS morphology. In our study, among the 5 "young" MDS with concomitant inherited RBC membrane defect, one case did not finally confirm MDS diagnosis by karyotype or NGS myeloid mutation analysis. This case fulfilled the diagnostic criteria for combined hereditary hemolytic anemia: DHS2, G6PD deficiency, spherocytosis, familial pseudohyperkalemia 2, with multi-locus mode of inheritance that justifies the clinical condition (splenomegaly, hemolytic crisis, ascites). Addition of genetic testing for hereditary anemias beside assessment of germline predisposing variants may allow a more precise diagnosis in controversial cases of "young" MDS. Disclosures Sanna: Janssen: Consultancy; Abbvie: Consultancy; Astra Zeneca: Consultancy. Iolascon: Celgene: Other: Advisory Board; Bluebird Bio: Other: Advisory Board. Santini: Menarini: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Geron: Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astex: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees.

Evaluation of the Main Regulators of Systemic Iron Homeostasis in Pyruvate Kinase Deficiency

Iron loading anemias are characterized by ineffective erythropoiesis and iron overload. This group of anemias includes thalassemia syndromes, congenital dyserythropoietic anemias (CDA), and some forms of congenital hemolytic anemias. Among them pyruvate kinase deficiency (PKD) has been shown to develop iron overload also in absence of transfusions suggesting dyserythropoietic features. Moreover, severe forms can be misdiagnosed as CDA due to bone marrow abnormalities and ineffective erythropoiesis further supporting this evidences. The hormone erythroferrone (hERFE) is produced by erythroblasts in response to erythropoietin (EPO), and acts by suppressing hepcidin, thereby increasing iron absorption and mobilisation for erythropoiesis demand. The ERFE-hepcidin axis seems to play a crucial role in the pathogenesis of these disorders; an increased erythroferrone release by immature erythroid cells results in hepcidin suppression and secondary iron overload that could finally results in ineffective erythropoiesis and anemia. To investigate the pathophysiological basis of iron overload in PKD, we analysed the levels of hERFE, EPO, hepcidin, and soluble transferrin receptor (sTFR) in a large group of 41 PKD patients equally distributed by gender, age and severity. The results were analysed in comparison with two groups of patients affected by hemolytic anemia with overt dyserythropoiesis (42 patients with CDA type II) and with congenital hemolytic anemia due to RBC membrane defects (51 patients with hereditary spherocytosis [HS]), respectively. Demographic, hematologic, and biochemical features of the three groups of patients are reported in the table. Among the PKD patients, 18/41 were &lt;18 yrs, median Hb level at the time of the study was 9.05g/dL (range 5.5-14.5), 12 underwent splenectomy, 28 ever received at least three transfusions their life, 14 of them transfusion dependent (&gt;6 tx/yrs). Mean ferritin levels at the time of the study were 546 ng/ml (range 59-4990), 15/41 patients requiring chelation therapy for iron overload developed also in absence of transfusions. As expected, CDAII patients showed decreased hepcidin levels (3.74 ng/mL; n.v. 17.25, P&lt;0.001) associated with increased erythropoietin (62.7 IU/L, n.v. 6.5, P=0.01) and hERFE (24.8 ng/mL, n.v. 1, P&lt;0.0001). On the contrary, HS showed increased hepcidin, with less marked increased of ERFE (9.9 ng/mL, P=0.02) and EPO (36.4IU/L, P=0.005). In PKD patients we observed decreased hepcidin levels (7.15 ng/mL, P=0.03)), increased hERFE (18ng/mL, P&lt;0.0001) and EPO (75.6 IU/L, P=0.009). Instead, sTFR was equally increased in the three groups of patients (Figure). Interestingly, by comparing the three groups of patients, PKD showed dyserythropoietic features as evidenced by the observation of intermediate values between HS and CDAII of hepcidin (P=0.007 PKD v CDAII and P=0.0002 PKD vs HS), hEFRE, and sTFR. This study provides the first analysis of the main regulators of systemic iron homeostasis in PK deficiency compared either with the model of a structural RBC defect (HS) or with the typical model of dyserythropoietic anemia with ineffective erythropoiesis, such as CDAII. These data provide evidence of the dyserythropoietic features of PK deficiency, underlining the need of accurate diagnosis and paving the way of novel therapeutic approaches in PK deficiency. Zaninoni A. and Russo R. equally contributed to the study Figure 1 Figure 1. Disclosures Fattizzo: Kira: Speakers Bureau; Alexion: Speakers Bureau; Novartis: Speakers Bureau; Momenta: Honoraria, Speakers Bureau; Annexon: Consultancy; Apellis: Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Barcellini: Incyte: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria. Iolascon: Bluebird Bio: Other: Advisory Board; Celgene: Other: Advisory Board. Bianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.