A randomized trial of blood donor iron repletion on red cell quality for transfusion and donor cognition and well-being.

Although altruistic regular blood donors are vital for the blood supply, many become iron deficient from donation-induced iron loss. The effects of blood donation-induced iron deficiency on red cell transfusion quality or donor cognition are unknown. In this double-blind, randomized trial, adult iron-deficient blood donors (n = 79; ferritin < 15 µg/L and zinc protoporphyrin >60 µMol/mol heme) who met donation qualifications were enrolled. A first standard blood donation was followed by the gold-standard measure for red cell storage quality: a 51-chromium posttransfusion red cell recovery study. Donors were then randomized to intravenous iron repletion (1 g low-molecular-weight iron dextran) or placebo. A second donation ∼5 months later was followed by another recovery study. Primary outcome was the within-subject change in posttransfusion recovery. The primary outcome measure of an ancillary study reported here was the National Institutes of Health Toolbox-derived uncorrected standard Cognition Fluid Composite Score. Overall, 983 donors were screened; 110 were iron-deficient, and of these, 39 were randomized to iron repletion and 40 to placebo. Red cell storage quality was unchanged by iron repletion: mean change in posttransfusion recovery was 1.6% (95% confidence interval -0.5 to 3.8) and -0.4% (-2.0 to 1.2) with and without iron, respectively. Iron repletion did not affect any cognition or well-being measures. These data provide evidence that current criteria for blood donation preserve red cell transfusion quality for the recipient and protect adult donors from measurable effects of blood donation-induced iron deficiency on cognition. This trial was registered at www.clinicaltrials.gov as NCT02889133 and NCT02990559.

Retention of functional mitochondria in mature red blood cells from patients with sickle cell disease.

Sickle cell disease (SCD) is an inherited blood disorder characterized by sickled red blood cells (RBCs), which are more sensitive to haemolysis and can contribute to disease pathophysiology. Although treatment of SCD can include RBC transfusion, patients with SCD have high rates of alloimmunization. We hypothesized that RBCs from patients with SCD have functionally active mitochondria and can elicit a type 1 interferon response. We evaluated blood samples from more than 100 patients with SCD and found elevated frequencies of mitochondria in reticulocytes and mature RBCs, as compared to healthy blood donors. The presence of mitochondria in mature RBCs was confirmed by flow cytometry, electron microscopy, and proteomic analysis. The mitochondria in mature RBCs were metabolically competent, as determined by enzymatic activities and elevated levels of mitochondria-derived metabolites. Metabolically-active mitochondria in RBCs may increase oxidative stress, which could facilitate and/or exacerbate SCD complications. Coculture of mitochondria-positive RBCs with neutrophils induced production of type 1 interferons, which are known to increase RBC alloimmunization rates. These data demonstrate that mitochondria retained in mature RBCs are functional and can elicit immune responses, suggesting that inappropriate retention of mitochondria in RBCs may play an underappreciated role in SCD complications and be an RBC alloimmunization risk factor.

Irradiation Causes Alterations of Polyamine, Purine, and Sulfur Metabolism in Red Blood Cells and Multiple Organs.

Investigating the metabolic effects of radiation is critical to understand the impact of radiotherapy, space travel, and exposure to environmental radiation. In patients undergoing hemopoietic stem cell transplantation, iron overload is a common risk factor for poor outcomes. However, no studies have interrogated the multiorgan effects of these treatments concurrently. Herein, we use a model that recapitulates transfusional iron overload, a condition often observed in chronically transfused patients. We applied an omics approach to investigate the impact of both the iron load and irradiation on the host metabolome. The results revealed dose-dependent effects of irradiation in the red blood cells, plasma, spleen, and liver energy and redox metabolism. Increases in polyamines and purine salvage metabolites were observed in organs with high oxygen consumption including the heart, kidneys, and brain. Irradiation also impacted the metabolism of the duodenum, colon, and stool, suggesting a potential effect on the microbiome. Iron infusion affected the response to radiation in the organs and blood, especially in erythrocyte polyamines and spleen antioxidant metabolism, and affected glucose, methionine, and glutathione systems and tryptophan metabolism in the liver, stool, and the brain. Together, the results suggest that radiation impacts metabolism on a multiorgan level with a significant interaction of the host iron status.

Red blood cell transfusion-induced non-transferrin-bound iron promotes Pseudomonas aeruginosa biofilms in human sera and mortality in catheterized mice.

Transfusion of storage-damaged red blood cells (RBCs) increases non-transferrin-bound iron (NTBI) levels in humans. This can potentially enhance virulence of microorganisms. In this study, Pseudomonas aeruginosa replication and biofilm production in vitro correlated with NTBI levels of transfused subjects (R2 = 0·80; P < 0·0001). Transfusion of stored RBCs into catheterized mice enhanced P. aeruginosa virulence and mortality in vivo, while pre-administration of apotransferrin reduced NTBI levels improving survival (69% vs 27% mortality; P < 0·05). These results suggest that longer RBC storage, by modulating the bioavailability of iron, may increase the risk of P. aeruginosa biofilm-related infections in transfused patients.

A New Murine Model of Primary Autoimmune Hemolytic Anemia (AIHA).

Loss of humoral tolerance to red blood cells (RBCs) can lead to autoimmune hemolytic anemia (AIHA), a severe, and sometimes fatal disease. Patients with AIHA present with pallor, fatigue, decreased hematocrit, and splenomegaly. While secondary AIHA is associated with lymphoproliferative disorders, infections, and more recently, as an adverse event secondary to cancer immunotherapy, the etiology of primary AIHA is unknown. Several therapeutic strategies are available; however, there are currently no licensed treatments for AIHA and few therapeutics offer treatment-free durable remission. Moreover, supportive care with RBC transfusions can be challenging as most autoantibodies are directed against ubiquitous RBC antigens; thus, virtually all RBC donor units are incompatible. Given the severity of AIHA and the lack of treatment options, understanding the cellular and molecular mechanisms that facilitate the breakdown in tolerance would provide insight into new therapeutics. Herein, we report a new murine model of primary AIHA that reflects the biology observed in patients with primary AIHA. Production of anti-erythrocyte autoantibodies correlated with sex and age, and led to RBC antigen modulation, complement fixation, and anemia, as determined by decreased hematocrit and hemoglobin values and increased reticulocytes in peripheral blood. Moreover, autoantibody-producing animals developed splenomegaly, with altered splenic architecture characterized by expanded white pulp areas and nearly diminished red pulp areas. Additional analysis suggested that compensatory extramedullary erythropoiesis occurred as there were increased frequencies of RBC progenitors detectable in the spleen. No significant correlations between AIHA onset and inflammatory status or microbiome were observed. To our knowledge, this is the first report of a murine model that replicates observations made in humans with idiopathic AIHA. Thus, this is a tractable murine model of AIHA that can serve as a platform to identify key cellular and molecular pathways that are compromised, thereby leading to autoantibody formation, as well as testing new therapeutics and management strategies.

A randomized double-blind controlled trial of convalescent plasma in adults with severe COVID-19.

BACKGROUNDAlthough convalescent plasma has been widely used to treat severe coronavirus disease 2019 (COVID-19), data from randomized controlled trials that support its efficacy are limited.METHODSWe conducted a randomized, double-blind, controlled trial among adults hospitalized with severe and critical COVID-19 at 5 sites in New York City (USA) and Rio de Janeiro (Brazil). Patients were randomized 2:1 to receive a single transfusion of either convalescent plasma or normal control plasma. The primary outcome was clinical status at 28 days following randomization, measured using an ordinal scale and analyzed using a proportional odds model in the intention-to-treat population.RESULTSOf 223 participants enrolled, 150 were randomized to receive convalescent plasma and 73 to receive normal control plasma. At 28 days, no significant improvement in the clinical scale was observed in participants randomized to convalescent plasma (OR 1.50, 95% confidence interval [CI] 0.83-2.68, P = 0.180). However, 28-day mortality was significantly lower in participants randomized to convalescent plasma versus control plasma (19/150 [12.6%] versus 18/73 [24.6%], OR 0.44, 95% CI 0.22-0.91, P = 0.034). The median titer of anti-SARS-CoV-2 neutralizing antibody in infused convalescent plasma units was 1:160 (IQR 1:80-1:320). In a subset of nasopharyngeal swab samples from Brazil that underwent genomic sequencing, no evidence of neutralization-escape mutants was detected.CONCLUSIONIn adults hospitalized with severe COVID-19, use of convalescent plasma was not associated with significant improvement in day 28 clinical status. However, convalescent plasma was associated with significantly improved survival. A possible explanation is that survivors remained hospitalized at their baseline clinical status.TRIAL REGISTRATIONClinicalTrials.gov, NCT04359810.FUNDINGAmazon Foundation, Skoll Foundation.

Distinct antibody responses to SARS-CoV-2 in children and adults across the COVID-19 clinical spectrum.

Clinical manifestations of COVID-19 caused by the new coronavirus SARS-CoV-2 are associated with age1,2. Adults develop respiratory symptoms, which can progress to acute respiratory distress syndrome (ARDS) in the most severe form, while children are largely spared from respiratory illness but can develop a life-threatening multisystem inflammatory syndrome (MIS-C)3-5. Here, we show distinct antibody responses in children and adults after SARS-CoV-2 infection. Adult COVID-19 cohorts had anti-spike (S) IgG, IgM and IgA antibodies, as well as anti-nucleocapsid (N) IgG antibody, while children with and without MIS-C had reduced breadth of anti-SARS-CoV-2-specific antibodies, predominantly generating IgG antibodies specific for the S protein but not the N protein. Moreover, children with and without MIS-C had reduced neutralizing activity as compared to both adult COVID-19 cohorts, indicating a reduced protective serological response. These results suggest a distinct infection course and immune response in children independent of whether they develop MIS-C, with implications for developing age-targeted strategies for testing and protecting the population.

Antibody responses to SARS-CoV2 are distinct in children with MIS-C compared to adults with COVID-19.

Clinical manifestations of COVID-19 caused by the novel coronavirus SARS-CoV-2 are associated with age. While children are largely spared from severe respiratory disease, they can present with a SARS-CoV-2-associated multisystem inflammatory syndrome (MIS-C) similar to Kawasaki's disease. Here, we show distinct antibody (Ab) responses in children with MIS-C compared to adults with severe COVID-19 causing acute respiratory distress syndrome (ARDS), and those who recovered from mild disease. There was a reduced breadth and specificity of anti-SARS-CoV-2-specific antibodies in MIS-C patients compared to the COVID patient groups; MIS-C predominantly generated IgG Abs specific for the Spike (S) protein but not for the nucleocapsid (N) protein, while both COVID-19 cohorts had anti-S IgG, IgM and IgA Abs, as well as anti-N IgG Abs. Moreover, MIS-C patients had reduced neutralizing activity compared to COVID-19 cohorts, indicating a reduced protective serological response. These results suggest a distinct infection course and immune response in children and adults who develop severe disease, with implications for optimizing treatments based on symptom and age.

Donor glucose-6-phosphate dehydrogenase deficiency decreases blood quality for transfusion.

BACKGROUNDGlucose-6-phosphate dehydrogenase (G6PD) deficiency decreases the ability of red blood cells (RBCs) to withstand oxidative stress. Refrigerated storage of RBCs induces oxidative stress. We hypothesized that G6PD-deficient donor RBCs would have inferior storage quality for transfusion as compared with G6PD-normal RBCs.METHODSMale volunteers were screened for G6PD deficiency; 27 control and 10 G6PD-deficient volunteers each donated 1 RBC unit. After 42 days of refrigerated storage, autologous 51-chromium 24-hour posttransfusion RBC recovery (PTR) studies were performed. Metabolomics analyses of these RBC units were also performed.RESULTSThe mean 24-hour PTR for G6PD-deficient subjects was 78.5% ± 8.4% (mean ± SD), which was significantly lower than that for G6PD-normal RBCs (85.3% ± 3.2%; P = 0.0009). None of the G6PD-normal volunteers (0/27) and 3 G6PD-deficient volunteers (3/10) had PTR results below 75%, a key FDA acceptability criterion for stored donor RBCs. As expected, fresh G6PD-deficient RBCs demonstrated defects in the oxidative phase of the pentose phosphate pathway. During refrigerated storage, G6PD-deficient RBCs demonstrated increased glycolysis, impaired glutathione homeostasis, and increased purine oxidation, as compared with G6PD-normal RBCs. In addition, there were significant correlations between PTR and specific metabolites in these pathways.CONCLUSIONBased on current FDA criteria, RBCs from G6PD-deficient donors would not meet the requirements for storage quality. Metabolomics assessment identified markers of PTR and G6PD deficiency (e.g., pyruvate/lactate ratios), along with potential compensatory pathways that could be leveraged to ameliorate the metabolic needs of G6PD-deficient RBCs.TRIAL REGISTRATIONClinicalTrials.gov NCT04081272.FUNDINGThe Harold Amos Medical Faculty Development Program, Robert Wood Johnson Foundation grant 71590, the National Blood Foundation, NIH grant UL1 TR000040, the Webb-Waring Early Career Award 2017 by the Boettcher Foundation, and National Heart, Lung, and Blood Institute grants R01HL14644 and R01HL148151.

Transfusional iron overload and intravenous iron infusions modify the mouse gut microbiota similarly to dietary iron.

Iron is essential for both microorganisms and their hosts. Although effects of dietary iron on gut microbiota have been described, the effect of systemic iron administration has yet to be explored. Here, we show that dietary iron, intravenous iron administration, and chronic transfusion in mice increase the availability of iron in the gut. These iron interventions have consistent and reproducible effects on the murine gut microbiota; specifically, relative abundance of the Parabacteroides and Lactobacillus genera negatively correlate with increased iron stores, whereas members of the Clostridia class positively correlate with iron stores regardless of the route of iron administration. Iron levels also affected microbial metabolites, in general, and indoles, in particular, circulating in host plasma and in stool pellets. Taken together, these results suggest that by shifting the balance of the microbiota, clinical interventions that affect iron status have the potential to alter biologically relevant microbial metabolites in the host.

Reexamination of the chromium-51-labeled posttransfusion red blood cell recovery method.

BACKGROUND: The chromium-51-labeled posttransfusion recovery (PTR) study has been the gold-standard test for assessing red blood cell (RBC) quality. Despite guiding RBC storage development for decades, it has several potential sources for error. METHODS: Four healthy adult volunteers each donated an autologous, leukoreduced RBC unit, aliquots were radiolabeled with technetium-99m after 1 and 6 weeks of storage, and then infused. Subjects were imaged by single-photon-emission computed tomography immediately and 4 hours after infusion. Additionally, from subjects described in a previously published study, adenosine triphosphate levels in transfusates infused into 52 healthy volunteers randomized to a single autologous, leukoreduced, RBC transfusion after 1, 2, 3, 4, 5, or 6 weeks of storage were correlated with PTR and laboratory parameters of hemolysis. RESULTS: Evidence from one subject imaged after infusion of technetium-99m-labeled RBCs suggests that, in some individuals, RBCs may be temporarily sequestered in the liver and spleen immediately following transfusion and then subsequently released back into circulation; this could be one source of error leading to PTR results that may not accurately predict the true quantity of RBCs cleared by intra- and/or extravascular hemolysis. Indeed, adenosine triphosphate levels in the transfusates correlated more robustly with measures of extravascular hemolysis in vivo (e.g., serum iron, indirect bilirubin, non-transferrin-bound iron) than with PTR results or measures of intravascular hemolysis (e.g., plasma free hemoglobin). CONCLUSIONS: Sources of measurement error are inherent in the chromium-51 PTR method. Transfusion of an entire unlabeled RBC unit, followed by quantifying extravascular hemolysis markers, may more accurately measure true posttransfusion RBC recovery.

Defective activation of the MAPK/ERK pathway, leading to PARP1 and DNMT1 dysregulation, is a common defect in IgA nephropathy and Henoch-Schönlein purpura.

Studies on IgA nephropathy (IgAN) have identified, through GWAS, linkage analysis, and pathway scanning, molecular defects in familial and sporadic IgAN patients. In our previous study, we identified a novel variant in the SPRY2 gene that segregates with the disease in one large family. The functional characterization of this variant led us to discover that the MAPK/ERK pathway was defective not only in this family, but also in two sporadic IgAN patients wild type for SPRY2. In the present study, we have deepened the molecular analysis of the MAPK/ERK pathway and extended our evaluation to a larger cohort of sporadic patients and to one additional family. We found that the ERK pathway is defective in IgAN patients and in patients affected by another IgA-mediated disorder, Henoch-Schönlein purpura (HSP). Furthermore, we found that two other proteins, PARP1 and DNMT1, respectively involved in DNA repair and in antibody class switching and methylation maintenance duties, were critically downregulated in IgAN and HSP patients. This study opens up the possibility that defective ERK activation, in some patients, leads to PARP1 and DNMT1 downregulation suggesting that IgAN could be the consequence of a dysregulated epigenetic maintenance leading to the upregulation of several genes. In particular, PARP1 could be used as a potential biomarker for the disease.

Annotating pathogenic non-coding variants in genic regions.

Identifying the underlying causes of disease requires accurate interpretation of genetic variants. Current methods ineffectively capture pathogenic non-coding variants in genic regions, resulting in overlooking synonymous and intronic variants when searching for disease risk. Here we present the Transcript-inferred Pathogenicity (TraP) score, which uses sequence context alterations to reliably identify non-coding variation that causes disease. High TraP scores single out extremely rare variants with lower minor allele frequencies than missense variants. TraP accurately distinguishes known pathogenic and benign variants in synonymous (AUC = 0.88) and intronic (AUC = 0.83) public datasets, dismissing benign variants with exceptionally high specificity. TraP analysis of 843 exomes from epilepsy family trios identifies synonymous variants in known epilepsy genes, thus pinpointing risk factors of disease from non-coding sequence data. TraP outperforms leading methods in identifying non-coding variants that are pathogenic and is therefore a valuable tool for use in gene discovery and the interpretation of personal genomes.While non-coding synonymous and intronic variants are often not under strong selective constraint, they can be pathogenic through affecting splicing or transcription. Here, the authors develop a score that uses sequence context alterations to predict pathogenicity of synonymous and non-coding genetic variants, and provide a web server of pre-computed scores.

Prolonged red cell storage before transfusion increases extravascular hemolysis.

BACKGROUND: Some countries have limited the maximum allowable storage duration for red cells to 5 weeks before transfusion. In the US, red blood cells can be stored for up to 6 weeks, but randomized trials have not assessed the effects of this final week of storage on clinical outcomes. METHODS: Sixty healthy adult volunteers were randomized to a single standard, autologous, leukoreduced, packed red cell transfusion after 1, 2, 3, 4, 5, or 6 weeks of storage (n = 10 per group). 51-Chromium posttransfusion red cell recovery studies were performed and laboratory parameters measured before and at defined times after transfusion. RESULTS: Extravascular hemolysis after transfusion progressively increased with increasing storage time (P < 0.001 for linear trend in the AUC of serum indirect bilirubin and iron levels). Longer storage duration was associated with decreasing posttransfusion red cell recovery (P = 0.002), decreasing elevations in hematocrit (P = 0.02), and increasing serum ferritin (P < 0.0001). After 6 weeks of refrigerated storage, transfusion was followed by increases in AUC for serum iron (P < 0.01), transferrin saturation (P < 0.001), and nontransferrin-bound iron (P < 0.001) as compared with transfusion after 1 to 5 weeks of storage. CONCLUSIONS: After 6 weeks of refrigerated storage, transfusion of autologous red cells to healthy human volunteers increased extravascular hemolysis, saturated serum transferrin, and produced circulating nontransferrin-bound iron. These outcomes, associated with increased risks of harm, provide evidence that the maximal allowable red cell storage duration should be reduced to the minimum sustainable by the blood supply, with 35 days as an attainable goal.REGISTRATION. ClinicalTrials.gov NCT02087514. FUNDING: NIH grant HL115557 and UL1 TR000040.

A SPRY2 mutation leading to MAPK/ERK pathway inhibition is associated with an autosomal dominant form of IgA nephropathy.

IgA nephropathy (IgAN) represents the most common primary glomerulonephritis worldwide with a prevalence of 25-50% among patients with primary glomerulopathies. In ~5-10% of the patients the disease segregates with an autosomal dominant (AD) pattern. Association studies identified loci on chromosomes 1q32, 6p21, 8p23, 17p13, 22q12, whereas classical linkage studies on AD families identified loci on chromosomes 2q36, 4q26-31, 6q22, 17q12-22. We have studied a large Sicilian family where IgAN segregates with an AD transmission. To identify the causal gene, the exomes of two affected and one unaffected individual have been sequenced. From the bioinformatics analysis a p.(Arg119Trp) variant in the SPRY2 gene was identified as the probable disease-causing mutation. Moreover, functional characterization of this variant showed that it is responsible for the inhibition of the MAPK/ERK1/2 pathway. The same effect was observed in two sporadic IgAN patients carriers of wild-type SPRY2, suggesting that downregulation of the MAPK/ERK1/2 pathway represents a common mechanism leading to IgAN.

T cell repertoire in DQ5-positive MuSK-positive myasthenia gravis patients.

Myasthenia gravis (MG) is a prototypical antibody-mediated disease characterized by muscle weakness and fatigability. Serum antibodies to the acetylcholine receptor and muscle-specific tyrosine kinase receptor (MuSK) are found in about 85% and 8% of patients respectively. We have previously shown that more than 70% of MG patients with MuSK antibodies share the HLA DQ5 allele. The aim of the present study was to analyze the T cell receptor (TCR) repertoire specific for recombinant human MuSK protein. We used the CDR3 TRBV-TRBJ spectratyping (immunoscope) to analyze the T cell response to MuSK from 13 DQ5+ MuSK-MG patients and from 7 controls (six DQ5+ MuSK negative subjects and one DQ5- DQ3+ MuSK positive patient). DQ5+ MuSK-MG patients but not controls used a restricted set of TCR VJ rearrangements in response to MuSK stimulation. One semiprivate (TRBV29-TRBJ2.5) rearrangement was found in 5/13 patients, while 4 other semiprivate (one in TRBV28-TRBJ2.1 and in TRBV3-TRBJ1.2, and two in TRBV28-TRBJ1.2) rearrangements were differently shared by 4/13 patients each and were absent in controls. When we sequenced the TRBV29-TRBJ2.5 rearrangement, we obtained 26 different sequences of the expected 130 bp length from 117 samples of the 5 positive patients: two common motifs GXGQET/TEHQET were shared in 4 patients as semiprivate motifs. Thus, the MuSK-specific T-cell response appears to be restricted in DQ5+ MuSK-MG patients, with a semiprivate repertoire including a common motif of TRBV29. This oligoclonal restriction of T cells will allow the identification of immunodominant epitopes in the antigen, providing therefore new tools for diagnosis and targeted therapy.

PTPN22 and myasthenia gravis: replication in an Italian population and meta-analysis of literature data.

Polymorphisms in PTPN22 are associated with many autoimmune diseases; while rs2476601 is supposed to play a major role, other experimental data suggest that rs2488457 may be even more important. Results in myasthenia gravis are controversial. In 356 Italian myasthenic patients and 439 controls genotyped for both polymorphisms, we found that rs2476601 was not associated with myasthenia, presence of autoantibodies, thymus pathology, sex or onset age unlike previous studies on other European populations (confirmed by the present meta-analysis). On the other hand, while rs2488457 was not associated with myasthenia or thymus pathology, we found a correlation of rs2488457 with low autoantibody titers and a trend of association with a less severe disease. Both polymorphisms were in tight linkage disequilibrium in controls, not in patients. Our results suggest that SNPs in this gene different from rs2476601, and/or epigenetic interactions, could play a greater role.