Characterization of a novel mouse platelet transfusion model.

BACKGROUND AND OBJECTIVES: Platelet transfusions are increasing with medical advances. Based on FDA criteria, platelet units are assessed by in vitro measures; however, it is not known how platelet processing and storage duration affect function in vivo. Our study's aim was to develop a novel platelet transfusion model stored in mouse plasma that meets FDA criteria adapted to mice, and transfused fresh and stored platelets are detectable in clots in vivo. STUDY DESIGN AND METHODS: Platelet units stored in mouse plasma were prepared using a modified platelet-rich plasma (PRP) collection protocol. Characteristics of fresh and stored units, including pH, cell count, in vitro measures of activity, including activation and aggregation, and post-transfusion recovery (PTR), were determined. Lastly, a tail transection assay was conducted using mice transfused with fresh or stored units, and transfused platelets were identified by confocal imaging. RESULTS: Platelet units had acceptable platelet and white cell counts and were negative for bacterial contamination. Fresh and 1-day stored units had acceptable pH; the platelets were activatable by thrombin and adenosine diphosphate, agreeable with thrombin, had acceptable PTR, and were present in vivo in clots of recipients after tail transection. In contrast, 2-day stored units had clinically unacceptable quality. CONCLUSION: We developed mouse platelets for transfusion analogous to human platelet units using a modified PRP collection protocol with maximum storage of 1 day for an 'old' unit. This provides a powerful tool to test how process modifications and storage conditions affect transfused platelet function in vivo.

Characterization of a Novel Mouse Platelet Transfusion Model.

BACKGROUND: Platelet transfusions are increasing with advances in medical care. Based on FDA criteria, platelet units are assessed by in vitro measures; however, it is not known how platelet processing and storage duration affect function in vivo. To address this, we developed a novel platelet transfusion model that meets FDA criteria adapted to mice, and transfused fresh and stored platelets are detected in clots in vivo. STUDY DESIGN AND METHODS: Platelet units stored in mouse plasma were prepared using a modified platelet rich plasma collection protocol. Characteristics of fresh and stored units, including pH, cell count, in vitro measures of activity, including activation and aggregation, and post-transfusion recovery (PTR), were determined. Lastly, a tail transection assay was conducted using mice transfused with fresh or stored units, and transfused platelets were identified by confocal imaging. RESULTS: Platelet units had acceptable platelet and white cell counts and were negative for bacterial contamination. Fresh and 1-day stored units had acceptable pH; the platelets were activatable by thrombin and ADP, aggregable with thrombin, had acceptable PTR, and were present in vivo in clots of recipients after tail transection. In contrast, 2-day stored units had clinically unacceptable quality. DISCUSSION: We developed mouse platelets for transfusion analogous to human platelet units using a modified platelet rich plasma collection protocol with maximum storage of 1 day for an "old" unit. This provides a powerful tool to test how process modifications and storage conditions affect transfused platelet function in vivo.

Low-Dose Dietary Fish Oil Improves RBC Deformability without Improving Post-Transfusion Recovery in Mice.

Long-chain polyunsaturated fatty acids (LC-PUFAs) are important modulators of red blood cell (RBC) rheology. Dietary LC-PUFAs are readily incorporated into the RBC membrane, improving RBC deformability, fluidity, and hydration. Female C57BL/6J mice consumed diets containing increasing amounts of fish oil (FO) ad libitum for 8 weeks. RBC deformability, filterability, and post-transfusion recovery (PTR) were evaluated before and after cold storage. Lipidomics and lipid peroxidation markers were evaluated in fresh and stored RBCs. High-dose dietary FO (50%, 100%) was associated with a reduction in RBC quality (i.e., in vivo lifespan, deformability, lipid peroxidation) along with a reduced 24 h PTR after cold storage. Low-dose dietary FO (6.25-12.5%) improved the filterability of fresh RBCs and reduced the lipid peroxidation of cold-stored RBCs. Although low doses of FO improved RBC deformability and reduced oxidative stress, no improvement was observed for the PTR of stored RBCs. The improvement in RBC deformability observed with low-dose FO supplementation could potentially benefit endurance athletes and patients with conditions resulting from reduced perfusion, such as peripheral vascular disease.

Transfusion medicine approaches for spontaneous intracerebral hemorrhage patients.

PURPOSE OF REVIEW: Spontaneous intracerebral hemorrhage (ICH) is the deadliest stroke subtype. Acute treatments necessitate rapid hemorrhage control to minimize secondary brain injury. Here, we discuss the overlap of transfusion medicine and acute ICH care relating to diagnostic testing and therapies relevant for coagulopathy reversal and secondary brain injury prevention. RECENT FINDINGS: Hematoma expansion (HE) is the largest contributor to poor outcomes after ICH. Conventional coagulation assays to diagnose coagulopathy after ICH does not predict HE. Given the testing limitations, empiric pragmatic hemorrhage control therapies have been trialed but have not improved ICH outcomes, with some therapies even causing harm. It is still unknown whether faster administration of these therapies will improve outcomes. Alternative coagulation tests (e.g., viscoelastic hemostatic assays, amongst others) may identify coagulopathies relevant for HE, currently not diagnosed using conventional assays. This provides opportunities for rapid, targeted therapies. In parallel, ongoing work is investigating alternative treatments using transfusion-based or transfusion-sparing pharmacotherapies that can be implemented in hemorrhage control strategies after ICH. SUMMARY: Further work is needed to identify improved laboratory diagnostic approaches and transfusion medicine treatment strategies to prevent HE and optimize hemorrhage control in ICH patients, who appear particularly vulnerable to the impacts of transfusion medicine practices.

Deuterated Linoleic Acid Attenuates the RBC Storage Lesion in a Mouse Model of Poor RBC Storage.

Background: Long-chain polyunsaturated fatty acids (PUFAs) are important modulators of red blood cell (RBC) rheology. Dietary PUFAs are readily incorporated into the RBC membrane, improving RBC deformability, fluidity, and hydration. However, enriching the lipid membrane with PUFAs increases the potential for peroxidation in oxidative environments (e.g., refrigerated storage), resulting in membrane damage. Substitution of bis-allylic hydrogens with deuterium ions in PUFAs decreases hydrogen abstraction, thereby inhibiting peroxidation. If lipid peroxidation is a causal factor in the RBC storage lesion, incorporation of deuterated linoleic acid (DLA) into the RBC membrane should decrease lipid peroxidation, thereby improving RBC lifespan, deformability, filterability, and post-transfusion recovery (PTR) after cold storage. Study Design and Methods: Mice associated with good (C57BL/6J) and poor (FVB) RBC storage quality received diets containing 11,11-D2-LA Ethyl Ester (1.0 g/100 g diet; deuterated linoleic acid) or non-deuterated LA Ethyl Ester (control) for 8 weeks. Deformability, filterability, lipidomics, and lipid peroxidation markers were evaluated in fresh and stored RBCs. Results: DLA was incorporated into RBC membranes in both mouse strains. DLA diet decreased lipid peroxidation (malondialdehyde) by 25.4 and 31% percent in C57 mice and 12.9 and 79.9% in FVB mice before and after cold storage, respectively. In FVB, but not C57 mice, deformability filterability, and post-transfusion recovery were significantly improved. Discussion: In a mouse model of poor RBC storage, with elevated reactive oxygen species production, DLA attenuated lipid peroxidation and significantly improved RBC storage quality.

Hematologic and systemic metabolic alterations due to Mediterranean class II G6PD deficiency in mice.

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is the single most common enzymopathy, present in approximately 400 million humans (approximately 5%). Its prevalence is hypothesized to be due to conferring resistance to malaria. However, G6PD deficiency also results in hemolytic sequelae from oxidant stress. Moreover, G6PD deficiency is associated with kidney disease, diabetes, pulmonary hypertension, immunological defects, and neurodegenerative diseases. To date, the only available mouse models have decreased levels of WT stable G6PD caused by promoter mutations. However, human G6PD mutations are missense mutations that result in decreased enzymatic stability. As such, this results in very low activity in red blood cells (RBCs) that cannot synthesize new protein. To generate a more accurate model, the human sequence for a severe form of G6PD deficiency, Med(-), was knocked into the murine G6PD locus. As predicted, G6PD levels were extremely low in RBCs, and deficient mice had increased hemolytic sequelae to oxidant stress. Nonerythroid organs had metabolic changes consistent with mild G6PD deficiency, consistent with what has been observed in humans. Juxtaposition of G6PD-deficient and WT mice revealed altered lipid metabolism in multiple organ systems. Together, these findings both establish a mouse model of G6PD deficiency that more accurately reflects human G6PD deficiency and advance our basic understanding of altered metabolism in this setting.

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.

Zika virus and its implications on cord blood banking and transplantation.

Umbilical cord blood is an important cellular therapy product used for hematopoietic stem cell transplantation, but the US Food and Drug Administration guidance regarding donor screening to reduce the risk of Zika transmission has decreased the number of licensed, eligible cord blood units available for transplantation. There is a crucial need for updated travel risk assessment for Zika virus transmission, validated screening tests for Zika virus in umbilical cord blood, and further research on Zika virus transmissibility due to umbilical cord blood products to ensure that umbilical cord blood and related tissues are safe and available for transplantation.

A Drosophila model of Fragile X syndrome exhibits defects in phagocytosis by innate immune cells.

Fragile X syndrome, the most common known monogenic cause of autism, results from the loss of FMR1, a conserved, ubiquitously expressed RNA-binding protein. Recent evidence suggests that Fragile X syndrome and other types of autism are associated with immune system defects. We found that Drosophila melanogaster Fmr1 mutants exhibit increased sensitivity to bacterial infection and decreased phagocytosis of bacteria by systemic immune cells. Using tissue-specific RNAi-mediated knockdown, we showed that Fmr1 plays a cell-autonomous role in the phagocytosis of bacteria. Fmr1 mutants also exhibit delays in two processes that require phagocytosis by glial cells, the immune cells in the brain: neuronal clearance after injury in adults and the development of the mushroom body, a brain structure required for learning and memory. Delayed neuronal clearance is associated with reduced recruitment of activated glia to the site of injury. These results suggest a previously unrecognized role for Fmr1 in regulating the activation of phagocytic immune cells both in the body and the brain.

period-Regulated Feeding Behavior and TOR Signaling Modulate Survival of Infection.

Most metazoans undergo dynamic, circadian-regulated changes in behavior and physiology. Currently, it is unknown how circadian-regulated behavior impacts immunity against infection. Two broad categories of defense against bacterial infection are resistance, control of microbial growth, and tolerance, control of the pathogenic effects of infection. Our study of behaviorally arrhythmic Drosophila circadian period mutants identified a novel link between nutrient intake and tolerance of infection with B. cepacia, a bacterial pathogen of rising importance in hospital-acquired infections. We found that infection tolerance in wild-type animals is stimulated by acute exposure to dietary glucose and amino acids. Glucose-stimulated tolerance was induced by feeding or direct injection; injections revealed a narrow window for glucose-stimulated tolerance. In contrast, amino acids stimulated tolerance only when ingested. We investigated the role of a known amino-acid-sensing pathway, the TOR (Target of Rapamycin) pathway, in immunity. TORC1 is circadian regulated and inhibition of TORC1 decreased resistance, as in vertebrates. Surprisingly, inhibition of the less well-characterized TOR complex 2 (TORC2) dramatically increased survival, through both resistance and tolerance mechanisms. This work suggests that dietary intake on the day of infection by B. cepacia can make a significant difference in long-term survival. We further demonstrate that TOR signaling mediates both resistance and tolerance of infection and identify TORC2 as a novel potential therapeutic target for increasing survival of infection.

The circadian clock protein timeless regulates phagocytosis of bacteria in Drosophila.

Survival of bacterial infection is the result of complex host-pathogen interactions. An often-overlooked aspect of these interactions is the circadian state of the host. Previously, we demonstrated that Drosophila mutants lacking the circadian regulatory proteins Timeless (Tim) and Period (Per) are sensitive to infection by S. pneumoniae. Sensitivity to infection can be mediated either by changes in resistance (control of microbial load) or tolerance (endurance of the pathogenic effects of infection). Here we show that Tim regulates resistance against both S. pneumoniae and S. marcescens. We set out to characterize and identify the underlying mechanism of resistance that is circadian-regulated. Using S. pneumoniae, we found that resistance oscillates daily in adult wild-type flies and that these oscillations are absent in Tim mutants. Drosophila have at least three main resistance mechanisms to kill high levels of bacteria in their hemolymph: melanization, antimicrobial peptides, and phagocytosis. We found that melanization is not circadian-regulated. We further found that basal levels of AMP gene expression exhibit time-of-day oscillations but that these are Tim-independent; moreover, infection-induced AMP gene expression is not circadian-regulated. We then show that phagocytosis is circadian-regulated. Wild-type flies exhibit up-regulated phagocytic activity at night; Tim mutants have normal phagocytic activity during the day but lack this night-time peak. Tim appears to regulate an upstream event in phagocytosis, such as bacterial recognition or activation of phagocytic hemocytes. Interestingly, inhibition of phagocytosis in wild type flies results in survival kinetics similar to Tim mutants after infection with S. pneumoniae. Taken together, these results suggest that loss of circadian oscillation of a specific immune function (phagocytosis) can have significant effects on long-term survival of infection.

FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism.

FMRP loss of function causes Fragile X syndrome (FXS) and autistic features. FMRP is a polyribosome-associated neuronal RNA-binding protein, suggesting that it plays a key role in regulating neuronal translation, but there has been little consensus regarding either its RNA targets or mechanism of action. Here, we use high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation (HITS-CLIP) to identify FMRP interactions with mouse brain polyribosomal mRNAs. FMRP interacts with the coding region of transcripts encoding pre- and postsynaptic proteins and transcripts implicated in autism spectrum disorders (ASD). We developed a brain polyribosome-programmed translation system, revealing that FMRP reversibly stalls ribosomes specifically on its target mRNAs. Our results suggest that loss of a translational brake on the synthesis of a subset of synaptic proteins contributes to FXS. In addition, they provide insight into the molecular basis of the cognitive and allied defects in FXS and ASD and suggest multiple targets for clinical intervention.

A mouse model of the human Fragile X syndrome I304N mutation.

The mental retardation, autistic features, and behavioral abnormalities characteristic of the Fragile X mental retardation syndrome result from the loss of function of the RNA-binding protein FMRP. The disease is usually caused by a triplet repeat expansion in the 5'UTR of the FMR1 gene. This leads to loss of function through transcriptional gene silencing, pointing to a key function for FMRP, but precluding genetic identification of critical activities within the protein. Moreover, antisense transcripts (FMR4, ASFMR1) in the same locus have been reported to be silenced by the repeat expansion. Missense mutations offer one means of confirming a central role for FMRP in the disease, but to date, only a single such patient has been described. This patient harbors an isoleucine to asparagine mutation (I304N) in the second FMRP KH-type RNA-binding domain, however, this single case report was complicated because the patient harbored a superimposed familial liver disease. To address these issues, we have generated a new Fragile X Syndrome mouse model in which the endogenous Fmr1 gene harbors the I304N mutation. These mice phenocopy the symptoms of Fragile X Syndrome in the existing Fmr1-null mouse, as assessed by testicular size, behavioral phenotyping, and electrophysiological assays of synaptic plasticity. I304N FMRP retains some functions, but has specifically lost RNA binding and polyribosome association; moreover, levels of the mutant protein are markedly reduced in the brain specifically at a time when synapses are forming postnatally. These data suggest that loss of FMRP function, particularly in KH2-mediated RNA binding and in synaptic plasticity, play critical roles in pathogenesis of the Fragile X Syndrome and establish a new model for studying the disorder.