Retinoid X receptor promotes hematopoietic stem cell fitness and quiescence and preserves hematopoietic homeostasis.

Hematopoietic stem cells (HSCs) balance self-renewal and differentiation to maintain hematopoietic fitness throughout life. In steady-state conditions, HSC exhaustion is prevented by the maintenance of most HSCs in a quiescent state, with cells entering the cell cycle only occasionally. HSC quiescence is regulated by retinoid and fatty-acid ligands of transcriptional factors of the nuclear retinoid X receptor (RXR) family. Herein, we show that dual deficiency for hematopoietic RXRα and RXRß induces HSC exhaustion, myeloid cell/megakaryocyte differentiation, and myeloproliferative-like disease. RXRα and RXRß maintain HSC quiescence, survival, and chromatin compaction; moreover, transcriptome changes in RXRα;RXRß-deficient HSCs include premature acquisition of an aging-like HSC signature, MYC pathway upregulation, and RNA intron retention. Fitness loss and associated RNA transcriptome and splicing alterations in RXRα;RXRß-deficient HSCs are prevented by Myc haploinsufficiency. Our study reveals the critical importance of RXRs for the maintenance of HSC fitness and their protection from premature aging.

Third-party virus-specific T cells for the treatment of double-stranded DNA viral reactivation and posttransplant lymphoproliferative disease after solid organ transplant.

Reactivation or primary infection with double-stranded DNA viruses is common in recipients of solid organ transplants (SOTs) and is associated with significant morbidity and mortality. Treatment with conventional antiviral medications is limited by toxicities, resistance, and a lack of effective options for adenovirus (ADV) and BK polyomavirus (BKPyV). Virus-specific T cells (VSTs) have been shown to be an effective treatment for infections with ADV, BKPyV, cytomegalovirus (CMV), and Epstein-Barr virus (EBV). Most of these studies have been conducted in stem cell recipients, and no large studies have been published in the SOT population to date. In this study, we report on the outcome of quadrivalent third-party VST infusions in 98 recipients of SOTs in the context of an open-label phase 2 trial. The 98 patients received a total of 181 infusions, with a median of 2 infusions per patient. The overall response rate was 45% for BKPyV, 65% for cytomegalovirus, 68% for ADV, and 61% for Epstein-Barr virus. Twenty percent of patients with posttransplant lymphoproliferative disorder had a complete response and 40% of patients had a partial response. All the VST infusions were well tolerated. We conclude that VSTs are safe and effective in the treatment of viral infections in SOT recipients.

In vitro quality parameters of whole blood-derived platelets pooled using two different platelet pooling sets and stored up to 7 days are similar.

BACKGROUND: Increasing the number of collections of whole blood-derived platelets (WBDP) and lengthening the allowable storage time may alleviate platelet (PLT) shortages. There is a need for new PLT pooling sets that can provide acceptable quality on Day 7 of storage. STUDY DESIGN AND METHODS: This pool-and-split study compared WBDP prepared using the platelet-rich plasma method with the novel IMUGARD WB PLT pooling set and a control pooling set. After pooling and filtration, PLT products were tested on Days 1, 5, and 7. Large volume delayed sampling (LVDS) cultures were taken on Day 2. RESULTS: The median postfiltration residual white blood cell (rWBC) content was 0.18 million per product (maximum 1.26 million; n = 69) with mean PLT recovery of 88.5 ± 2.8% for the new set and median 0.23 million (maximum 1.83 million) rWBC with 87.5 ± 2.5% recovery for the control. Day 5 mean pH22°C were 7.18 ± 0.12 and 7.13 ± 0.10 for the new and control set, respectively. Day 5 in vitro quality parameters were within 20% between the two pooling sets. The new set Day 7 pH22°C was acceptable (7.07 ± 0.17, 100% ≥ 6.3), and most parameters were within 20% of Day 5 values. CONCLUSION: WBDP quality for the new pooling set is acceptable across a battery of in vitro tests when stored up to 7 days and meets FDA regulatory criteria. The quality parameters were similar between the new pooling set and the control set on Day 5. This new set is compatible with LVDS.

Structure-Activity Relationship Analysis of Rhosin, a RhoA GTPase Inhibitor, Reveals a New Class of Antiplatelet Agents.

Current antiplatelet therapies have several clinical complications and are mostly irreversible in terms of suppressing platelet activity; hence, there is a need to develop improved therapeutic agents. Previous studies have implicated RhoA in platelet activation. Here, we further characterized the lead RhoA inhibitor, Rhosin/G04, in platelet function and present structure-activity relationship (SAR) analysis. A screening for Rhosin/G04 analogs in our chemical library by similarity and substructure searches revealed compounds that showed enhanced antiplatelet activity and suppressed RhoA activity and signaling. A screening for Rhosin/G04 analogs in our chemical library using similarity and substructure searches revealed compounds that showed enhanced antiplatelet activity and suppressed RhoA activity and signaling. SAR analysis revealed that the active compounds have a quinoline group optimally attached to the hydrazine at the 4-position and halogen substituents at the 7- or 8-position. Having indole, methylphenyl, or dichloro-phenyl substituents led to better potency. Rhosin/G04 contains a pair of enantiomers, and S-G04 is significantly more potent than R-G04 in inhibiting RhoA activation and platelet aggregation. Furthermore, the inhibitory effect is reversible, and S-G04 is capable of inhibiting diverse-agonist-stimulated platelet activation. This study identified a new generation of small-molecule RhoA inhibitors, including an enantiomer capable of broadly and reversibly modulating platelet activity.

Novel blood derived hemostatic agents for bleeding therapy and prophylaxis.

PURPOSE OF REVIEW: Hemorrhage is a major cause of preventable death in trauma and cancer. Trauma induced coagulopathy and cancer-associated endotheliopathy remain major therapeutic challenges. Early, aggressive administration of blood-derived products with hypothesized increased clotting potency has been proposed. A series of early- and late-phase clinical trials testing the safety and/or efficacy of lyophilized plasma and new forms of platelet products in humans have provided light on the future of alternative blood component therapies. This review intends to contextualize and provide a critical review of the information provided by these trials. RECENT FINDINGS: The beneficial effect of existing freeze-dried plasma products may not be as high as initially anticipated when tested in randomized, multicenter clinical trials. A next-generation freeze dried plasma product has shown safety in an early phase clinical trial and other freeze-dried plasma and spray-dried plasma with promising preclinical profiles are embarking in first-in-human trials. New platelet additive solutions and forms of cryopreservation or lyophilization of platelets with long-term shelf-life have demonstrated feasibility and logistical advantages. SUMMARY: Recent trials have confirmed logistical advantages of modified plasma and platelet products in the treatment or prophylaxis of bleeding. However, their postulated increased potency profile remains unconfirmed.

Yap1-Scribble polarization is required for hematopoietic stem cell division and fate.

Yap1 and its paralogue Taz largely control epithelial tissue growth. We have identified that hematopoietic stem cell (HSC) fitness response to stress depends on Yap1 and Taz. Deletion of Yap1 and Taz induces a loss of HSC quiescence, symmetric self-renewal ability, and renders HSC more vulnerable to serial myeloablative 5-fluorouracil treatment. This effect depends on the predominant cytosolic polarization of Yap1 through a PDZ domain-mediated interaction with the scaffold Scribble. Scribble and Yap1 coordinate to control cytoplasmic Cdc42 activity and HSC fate determination in vivo. Deletion of Scribble disrupts Yap1 copolarization with Cdc42 and decreases Cdc42 activity, resulting in increased self-renewing HSC with competitive reconstitution advantages. These data suggest that Scribble/Yap1 copolarization is indispensable for Cdc42-dependent activity on HSC asymmetric division and fate. The combined loss of Scribble, Yap1, and Taz results in transcriptional upregulation of Rac-specific guanine nucleotide exchange factors, Rac activation, and HSC fitness restoration. Scribble links Cdc42 and the cytosolic functions of the Hippo signaling cascade in HSC fate determination.

Bone marrow regeneration requires mitochondrial transfer from donor Cx43-expressing hematopoietic progenitors to stroma.

The fate of hematopoietic stem and progenitor cells (HSPC) is tightly regulated by their bone marrow (BM) microenvironment (ME). BM transplantation (BMT) frequently requires irradiation preconditioning to ablate endogenous hematopoietic cells. Whether the stromal ME is damaged and how it recovers after irradiation is unknown. We report that BM mesenchymal stromal cells (MSC) undergo massive damage to their mitochondrial function after irradiation. Donor healthy HSPC transfer functional mitochondria to the stromal ME, thus improving mitochondria activity in recipient MSC. Mitochondrial transfer to MSC is cell-contact dependent and mediated by HSPC connexin-43 (Cx43). Hematopoietic Cx43-deficient chimeric mice show reduced mitochondria transfer, which was rescued upon re-expression of Cx43 in HSPC or culture with isolated mitochondria from Cx43 deficient HSPCs. Increased intracellular adenosine triphosphate levels activate the purinergic receptor P2RX7 and lead to reduced activity of adenosine 5'-monophosphate-activated protein kinase (AMPK) in HSPC, dramatically increasing mitochondria transfer to BM MSC. Host stromal ME recovery and donor HSPC engraftment were augmented after mitochondria transfer. Deficiency of Cx43 delayed mesenchymal and osteogenic regeneration while in vivo AMPK inhibition increased stromal recovery. As a consequence, the hematopoietic compartment reconstitution was improved because of the recovery of the supportive stromal ME. Our findings demonstrate that healthy donor HSPC not only reconstitute the hematopoietic system after transplantation, but also support and induce the metabolic recovery of their irradiated, damaged ME via mitochondria transfer. Understanding the mechanisms regulating stromal recovery after myeloablative stress are of high clinical interest to optimize BMT procedures and underscore the importance of accessory, non-HSC to accelerate hematopoietic engraftment.

Antibodies against biotin-labeled red blood cells can shorten posttransfusion survival.

BACKGROUND: In hematologic and transfusion medicine research, measurement of red blood cell (RBC) in vivo kinetics must be safe and accurate. Recent reports indicate use of biotin-labeled RBC (BioRBC) to determine red cell survival (RCS) offers substantial advantages over 51 Cr and other labeling methods. Occasional induction of BioRBC antibodies has been reported. STUDY DESIGN AND METHODS: To investigate the causes and consequences of BioRBC immunization, we reexposed three previously immunized adults to BioRBC and evaluated the safety, antibody emergence, and RCS of BioRBC. RESULTS: BioRBC re-exposure caused an anamnestic increase of plasma BioRBC antibodies at 5-7 days; all were subclass IgG1 and neutralized by biotinylated albumin, thus indicating structural specificity for the biotin epitope. Concurrently, specific antibody binding to BioRBC was observed in each subject. As biotin label density increased, the proportion of BioRBC that bound increased antibody also increased; the latter was associated with proportional accelerated removal of BioRBC labeled at density 6 µg/mL. In contrast, only one of three subjects exhibited accelerated removal of BioRBC density 2 µg/mL. No adverse clinical or laboratory events were observed. Among three control subjects who did not develop BioRBC antibodies following initial BioRBC exposure, re-exposure induced neither antibody emergence nor accelerated BioRBC removal. DISCUSSION: We conclude re-exposure of immunized subjects to BioRBC can induce anamnestic antibody response that can cause an underestimation of RCS. To minimize chances of antibody induction and underestimation of RCS, we recommend an initial BioRBC exposure volume of ≤10 mL and label densities of ≤18 µg/mL.

Evaluation of amotosalen and UVA pathogen-reduced apheresis platelets after 7-day storage.

BACKGROUND: Amotosalen/UVA pathogen-reduced platelet components (PRPCs) with storage up to 7 days are standard of care in France, Switzerland, and Austria. PRPCs provide effective hemostasis with reduced risk of transfusion-transmitted infections and transfusion-associated graft versus host disease, reduced wastage and improved availability compared with 5-day-stored PCs. This study evaluated the potency of 7-day PRPCs by in vitro characterization and in vivo pharmacokinetic analysis of autologous PCs. STUDY DESIGN AND METHODS: The in vitro characteristics of 7-day-stored apheresis PRPCs suspended in 100% plasma or 65% platelet additive solution (PAS-3)/35% plasma, thrombin generation, and in vivo radiolabeled post-transfusion recovery and survival of 7-day-stored PRPCs suspended in 100% plasma were compared with either 7-day-stored or fresh autologous conventional platelets. RESULTS: PRPCs after 7 days of storage maintained pH, platelet dose, in vitro physiologic characteristics, and thrombin generation when compared to conventional 7-day PCs. In vivo, the mean post-transfusion survival was 151.4 ± 20.1 h for 7-day PRPCs in 100% plasma (Test) versus 209.6 ± 13.9 h for the fresh autologous platelets (Control), (T-ΔC: 72.3 ± 8.8%: 95% confidence interval [CI]: 68.5, 76.1) and mean 24-h post-transfusion recovery 37.6 ± 8.4% for Test versus 56.8 ± 9.2% for Control (T-ΔC: 66.2 ± 11.2%; 95% CI: 61.3, 71.1). DISCUSSION: PRPCs collected in both 100% plasma as well as 65% PAS-3/35% plasma and stored for 7 days retained in vitro physiologic characteristics. PRPCs stored in 100% plasma for 7 days retained in vivo survival. Lower in vivo post-radiolabeled autologous platelet recovery is consistent with reported reduced count increments for allogenic transfusion.

Characterization and first-in-human clinical dose-escalation safety evaluation of a next-gen human freeze-dried plasma.

BACKGROUND: Early plasma transfusion is life-saving for bleeding trauma patients. Freeze-dried plasma (FDP) provides unique formulation advantages for infusion in the prehospital setting. We describe characterization and clinical safety data of the first, next-generation FDP stored in plastic bags with rapid reconstitution. STUDY DESIGN AND METHODS: Coagulation and chemistry parameters on 155 pairs of fresh frozen plasma (FFP) and their derivative FDP units were compared. Next, a first-in-human, dose-escalation safety evaluation of FDP, involving 24 healthy volunteers who donated either whole blood or apheresis plasma to create autologous FDP, was performed in three dose cohorts (270, 540, and 810 ml) and adverse events (AEs) were monitored. Cohort 3 was randomized, double-blind with a cross-over arm that compared FDP versus FFP using descriptive analysis for AEs, coagulation, hematology, and chemistry parameters. RESULTS: FDP coagulation factors, clotting times, and product quality (pH, total protein, and osmolality) post-lyophilization were preserved. FDP infusions, of up to 810 ml per subject, were found to be safe and with no serious AEs (SAEs) related to FDP. The average time to reconstitute FDP was 67 s (range: 43-106). No differences in coagulation parameters or thrombin activation were detected in subjects infused with 810 ml of FDP compared with FFP. CONCLUSION: This first next-generation FDP product preserves the potency and safety of FFP in a novel rugged, compressible, plastic container, for rapid transfusion, allowing rapid access to plasma in resuscitation protocols for therapy in acute traumatic hemorrhage.

Freeze-dried platelets are a promising alternative in bleeding thrombocytopenic patients with hematological malignancies.

Thrombosomes are trehalose-stabilized, freeze-dried group O platelets with a 3-year shelf life. They can be stockpiled, rapidly reconstituted, and infused regardless of the recipient's blood type. Thrombosomes thus represent a potential alternative platelet transfusion strategy. The present study assessed the safety and potential early signals of efficacy of Thrombosomes in bleeding thrombocytopenic patients. We performed an open-label, phase 1 study of single doses of allogeneic Thrombosomes at three dose levels in three cohorts, each consisting of eight patients who had hematologic malignancies, thrombocytopenia, and bleeding. Adverse events, dose-limiting toxicities (DLTs), World Health Organization (WHO) bleeding scores, and hematology values were assessed. No DLTs were reported. The median age was 59 years (24-71). Most patients had AML (58%) or ALL (29%), followed by MDS (8%) and myeloproliferative neoplasm (4%). The WHO scores of 22 patients who were actively bleeding at a total of 27 sites at baseline either improved (n = 17 [63%]) or stabilized (n = 10 [37%]) through day 6. Twenty-four hours after infusion, 12 patients (50%) had a clinically significant platelet count increase. Of eight patients who received no platelet transfusions for 6 days after Thrombosomes infusion, 5 had a clinically significant increase in platelet count of ≥5000 platelets/µL and 2 had platelet count normalization. Thrombosomes doses up to 3.78 × 108 particles/kg demonstrated safety in 24 bleeding, thrombocytopenic patients with hematological malignancies. Thrombosomes may represent an alternative to conventional platelets to treat bleeding. A phase 2 clinical trial in a similar patient population is underway.

Osteopontin attenuates aging-associated phenotypes of hematopoietic stem cells.

Upon aging, hematopoietic stem cells (HSCs) undergo changes in function and structure, including skewing to myeloid lineages, lower reconstitution potential and loss of protein polarity. While stem cell intrinsic mechanisms are known to contribute to HSC aging, little is known on whether age-related changes in the bone marrow niche regulate HSC aging. Upon aging, the expression of osteopontin (OPN) in the murine bone marrow stroma is reduced. Exposure of young HSCs to an OPN knockout niche results in a decrease in engraftment, an increase in long-term HSC frequency and loss of stem cell polarity. Exposure of aged HSCs to thrombin-cleaved OPN attenuates aging of old HSCs, resulting in increased engraftment, decreased HSC frequency, increased stem cell polarity and a restored balance of lymphoid and myeloid cells in peripheral blood. Thus, our data suggest a critical role for reduced stroma-derived OPN for HSC aging and identify thrombin-cleaved OPN as a novel niche informed therapeutic approach for ameliorating HSC phenotypes associated with aging.

Antioxidant prevents clearance of hemostatically competent platelets after long-term cold storage.

BACKGROUND: Cold storage of platelets (PLTs) has the potential advantage of prolonging storage time while reducing posttransfusion infection given the decreased likelihood of bacterial outgrowth during storage and possibly beneficial effects in treating bleeding patients. However, cold storage reduces PLT survival through the induction of complex storage lesions, which are more accentuated when storage is prolonged. STUDY DESIGN AND METHODS: Whole blood-derived PLT-rich plasma concentrates from seven PLT pools (n = 5 donors per pool). PLT additive solution was added (67%/33% plasma) and the product was split into 50-mL bags. Split units were stored in the presence or absence of 1 mM of N-acetylcysteine (NAC) under agitation for up to 14 days at room temperature or in the cold and were analyzed for PLT activation, fibrinogen-dependent spreading, microparticle formation, mitochondrial respiratory activity, reactive oxygen species (ROS) generation, as well as in vivo survival and bleeding time correction in immunodeficient mice. RESULTS: Cold storage of PLTs for 7 days or longer induces significant PLT activation, cytoskeletal damage, impaired fibrinogen spreading, enhances mitochondrial metabolic decoupling and ROS generation, and increases macrophage-dependent phagocytosis and macrophage-independent clearance. Addition of NAC prevents PLT clearance and allows a correction of the prolonged bleeding time in thrombocytopenic, aspirin-treated, immunodeficient mice. CONCLUSIONS: Long-term cold storage induces mitochondrial uncoupling and increased proton leak and ROS generation. The resulting ROS is a crucial contributor to the increased macrophage-dependent and -independent clearance of functional PLTs and can be prevented by the antioxidant NAC in a magnesium-containing additive solution.

NF1 patient missense variants predict a role for ATM in modifying neurofibroma initiation.

In Neurofibromatosis type 1, NF1 gene mutations in Schwann cells (SC) drive benign plexiform neurofibroma (PNF), and no additional SC changes explain patient-to-patient variability in tumor number. Evidence from twin studies suggests that variable expressivity might be caused by unidentified modifier genes. Whole exome sequencing of SC and fibroblast DNA from the same resected PNFs confirmed biallelic SC NF1 mutations; non-NF1 somatic SC variants were variable and present at low read number. We identified frequent germline variants as possible neurofibroma modifier genes. Genes harboring variants were validated in two additional cohorts of NF1 patients and by variant burden test. Genes including CUBN, CELSR2, COL14A1, ATR and ATM also showed decreased gene expression in some neurofibromas. ATM-relevant DNA repair defects were also present in a subset of neurofibromas with ATM variants, and in some neurofibroma SC. Heterozygous ATM G2023R or homozygous S707P variants reduced ATM protein expression in heterologous cells. In mice, genetic Atm heterozygosity promoted Schwann cell precursor self-renewal and increased tumor formation in vivo, suggesting that ATM variants contribute to neurofibroma initiation. We identify germline variants, rare in the general population, overrepresented in NF1 patients with neurofibromas. ATM and other identified genes are candidate modifiers of PNF pathogenesis.

Hypoxic storage of red blood cells improves metabolism and post-transfusion recovery.

BACKGROUND: Blood transfusion is a lifesaving intervention for millions of recipients worldwide every year. Storing blood makes this possible but also promotes a series of alterations to the metabolism of the stored erythrocyte. It is unclear whether the metabolic storage lesion is correlated with clinically relevant outcomes and whether strategies aimed at improving the metabolic quality of stored units, such as hypoxic storage, ultimately improve performance in the transfused recipient. STUDY DESIGN AND METHODS: Twelve healthy donor volunteers were recruited in a two-arm cross-sectional study, in which each subject donated 2 units to be stored under standard (normoxic) or hypoxic conditions (Hemanext technology). End-of-storage measurements of hemolysis and autologous posttransfusion recovery (PTR) were correlated to metabolomics measurements at Days 0, 21, and 42. RESULTS: Hypoxic red blood cells (RBCs) showed superior PTR and comparable hemolysis to donor-paired standard units. Hypoxic storage improved energy and redox metabolism (glycolysis and 2,3-diphosphoglycerate), improved glutathione and methionine homeostasis, decreased purine oxidation and membrane lipid remodeling (free fatty acid levels, unsaturation and hydroxylation, acyl-carnitines). Intra- and extracellular metabolites in these pathways (including some dietary purines) showed significant correlations with PTR and hemolysis, though the degree of correlation was influenced by sulfur dioxide (SO2 ) levels. CONCLUSION: Hypoxic storage improves energy and redox metabolism of stored RBCs, which results in improved posttransfusion recoveries in healthy autologous recipients-a Food and Drug Administration gold standard of stored blood quality. In addition, we identified candidate metabolic predictors of PTR for RBCs stored under standard and hypoxic conditions.

Gap Junctions in the Bone Marrow Lympho-Hematopoietic Stem Cell Niche, Leukemia Progression, and Chemoresistance.

Abstract: The crosstalk between hematopoietic stem cells (HSC) and bone marrow (BM) microenvironment is critical for homeostasis and hematopoietic regeneration in response to blood formation emergencies after injury, and has been associated with leukemia transformation and progression. Intercellular signals by the BM stromal cells in the form of cell-bound or secreted factors, or by physical interaction, regulate HSC localization, maintenance, and differentiation within increasingly defined BM HSC niches. Gap junctions (GJ) are comprised of arrays of membrane embedded channels formed by connexin proteins, and control crucial signaling functions, including the transfer of ions, small metabolites, and organelles to adjacent cells which affect intracellular mechanisms of signaling and autophagy. This review will discuss the role of GJ in both normal and leukemic hematopoiesis, and highlight some of the most novel approaches that may improve the efficacy of cytotoxic drugs. Connexin GJ channels exert both cell-intrinsic and cell-extrinsic effects on HSC and BM stromal cells, involved in regenerative hematopoiesis after myelosuppression, and represent an alternative system of cell communication through a combination of electrical and metabolic coupling as well as organelle transfer in the HSC niche. GJ intercellular communication (GJIC) in the HSC niche improves cellular bioenergetics, and rejuvenates damaged recipient cells. Unfortunately, they can also support leukemia proliferation and survival by creating leukemic niches that provide GJIC dependent energy sources and facilitate chemoresistance and relapse. The emergence of new strategies to disrupt self-reinforcing malignant niches and intercellular organelle exchange in leukemic niches, while at the same time conserving normal hematopoietic GJIC function, could synergize the effect of chemotherapy drugs in eradicating minimal residual disease. An improved understanding of the molecular basis of connexin regulation in normal and leukemic hematopoiesis is warranted for the re-establishment of normal hematopoiesis after chemotherapy.

Future of platelet formulations with improved clotting profile: a short review on human safety and efficacy data.

BACKGROUND: Platelet (PLT) transfusion is a widely used therapy in treating or preventing bleeding and hemorrhage in patients with thrombocytopenia or trauma. Compared to the relative ease of PLT transfusion, current practice for PLT storage at room temperature (RT) for up to 5 to 7 days is inefficient, costly, wasteful, and relatively unsafe. STUDY DESIGN AND METHODS: This study was a review of major advances in PLT derivative products with improved hemostatic potential and safety feature. RESULTS: Recent progress in understanding the PLT activation and host clearance mechanisms has led to reassessments of current and new storage conditions that employ refrigeration and/or cryopreservation to overcome storage lesions and significantly extend shelf life of PLTs with reduced risk of pathogen contamination. DISCUSSION: It is anticipated that future PLT preservation involving cold, frozen, and/or pathogen reduction strategies in proper PLT additive solutions will enable longer term and safer PLT storage.

Towards increasing shelf life and haemostatic potency of stored platelet concentrates.

PURPOSE OF REVIEW: Platelet transfusion is a widely used therapy in treating or preventing bleeding and haemorrhage in patients with thrombocytopenia or trauma. Compared with the relative ease of platelet transfusion, current practice for the storage of platelets is inefficient, costly and relatively unsafe, with platelets stored at room temperature (RT) for upto 5-7 days. RECENT FINDINGS: During storage, especially at cold temperatures, platelets undergo progressive and deleterious changes, collectively termed the 'platelet storage lesion', which decrease their haemostatic function and posttransfusion survival. Recent progress in understanding platelet activation and host clearance mechanisms is leading to the consideration of both old and novel storage conditions that use refrigeration and/or cryopreservation to overcome various storage lesions and significantly extend platelet shelf-life with a reduced risk of pathogen contamination. SUMMARY: A review of the advantages and disadvantages of alternative methods for platelet storage is presented from both a clinical and biological perspective. It is anticipated that future platelet preservation involving cold, frozen and/or pathogen reduction strategies in a proper platelet additive solution will enable longer term and safer platelet storage.

Expansion of EPOR-negative macrophages besides erythroblasts by elevated EPOR signaling in erythrocytosis mouse models.

Activated erythropoietin (EPO) receptor (EPOR) signaling causes erythrocytosis. The important role of macrophages for the erythroid expansion and differentiation process has been reported, both in baseline and stress erythropoiesis. However, the significance of EPOR signaling for regulation of macrophages contributing to erythropoiesis has not been fully understood. Here we show that EPOR signaling activation quickly expands both erythrocytes and macrophages in vivo in mouse models of primary and secondary erythrocytosis. To mimic the chimeric condition and expansion of the disease clone in the polycythemia vera patients, we combined Cre-inducible Jak2V617F/+ allele with LysM-Cre allele which expresses in mature myeloid cells and some of the HSC/Ps (LysM-Cre;Jak2V617F/+ mice). We also generated inducible EPO-mediated secondary erythrocytosis models using Alb-Cre, Rosa26-loxP-stop-loxP-rtTA, and doxycycline inducible EPAS1-double point mutant (DPM) alleles (Alb-Cre;DPM mice). Both models developed a similar degree of erythrocytosis. Macrophages were also increased in both models without increase of major inflammatory cytokines and chemokines. EPO administration also quickly induced these macrophages in wild-type mice before observable erythrocytosis. These findings suggest that EPOR signaling activation could induce not only erythroid cell expansion, but also macrophages. Surprisingly, an in vivo genetic approach indicated that most of those macrophages do not express EPOR, but erythroid cells and macrophages contacted tightly with each other. Given the importance of the central macrophages as a niche for erythropoiesis, further elucidation of the EPOR signaling mediated-regulatory mechanisms underlying macrophage induction might reveal a potential therapeutic target for erythrocytosis.