Inhibition of RHOA activity preserves the survival and hemostasis function of long-term cold stored platelets.

Thrombocytopenic patients require platelet transfusion to prevent and stop hemorrhage. Cold storage of platelets results in complex molecular lesions including changes in membrane microdomains that are recognized by host macrophages and hepatocyte counter-receptors, resulting in phagocytosis and clearance upon transfusion. For this reason, platelets are stored at room temperature, a method that confers increased risk of bacterial contamination. By applying signaling analysis as well as genetic and pharmacological approaches, we identified that the cold induced activation of RHOA GTPase is causal for the major hallmarks of platelet cold storage lesions. RHOA deficiency renders murine platelets insensitive to cold storage induced damage, and pharmacological inhibition by a RHOA activation inhibitor, R-G04, can prevent the cold storage induced lesions. RHOA inhibition prevents myosin activation and clathrin-independent formation and internalization of lipid rafts enriched in active glycosyltransferases as well as abnormal distribution of GpIb. RHOA inhibition further prevents the metabolic reprogramming of cold induced storage lesions and allows the maintenance of glycolytic flux and mitochondrial dependent respiration. Importantly, human platelets transfused in mice after cold storage, in the presence of R-G04 or its more potent enantiomer S-G04, can circulate in vivo at similar levels as room-temperature stored platelets while retaining their hemostatic activity in vivo as assessed by bleeding time correction of aspirin-treated mice. Our studies provide a new mechanism based translational venue to prevent cold storage induced damage useful for human platelet transfusion in thrombocytopenic patients.

From Marrow to Bone and Fat: Exploring the Multifaceted Roles of Leptin Receptor Positive Bone Marrow Mesenchymal Stromal Cells.

The bone marrow (BM) stromal cell microenvironment contains non-hematopoietic stromal cells called mesenchymal stromal cells (MSCs). MSCs are plastic adherent, form CFU-Fs, and give rise to osteogenic, adipogenic, chondrogenic progenitors, and most importantly provide HSC niche factor chemokine C-X-C motif ligand 12 (CXCL12) and stem cell factor (SCF). Different authors have defined different markers for mouse MSC identification like PDGFR+Sca-1+ subsets, Nestin+, or LepR+ cells. Of these, the LepR+ cells are the major source of SCF and CXCL12 in the BM microenvironment and play a major role in HSC maintenance and hematopoiesis. LepR+ cells give rise to most of the bones and BM adipocytes, further regulating the microenvironment. In adult BM, LepR+ cells are quiescent but after fracture or irradiation, they proliferate and differentiate into mesenchymal lineage osteogenic, adipogenic and/or chondrogenic cells. They also play a crucial role in the steady-state hematopoiesis process, as well as hematopoietic regeneration and the homing of hematopoietic stem cells (HSCs) after myeloablative injury and/or HSC transplantation. They line the sinusoidal cavities, maintain the trabeculae formation, and provide the space for HSC homing and retention. However, the LepR+ cell subset is heterogeneous; some subsets have higher adipogenic potential, while others express osteollineage-biased genes. Different transcription factors like Early B cell factor 3 (EBF3) or RunX2 help maintain this balance between the self-renewing and committed states, whether osteogenic or adipogenic. The study of LepR+ MSCs holds immense promise for advancing our understanding of HSC biology, tissue regeneration, metabolic disorders, and immune responses. In this review, we will discuss the origin of the BM resident LepR+ cells, different subtypes, and the role of LepR+ cells in maintaining hematopoiesis, osteogenesis, and BM adipogenesis following their multifaceted impact.

A toxicology study of Csf2ra complementation and pulmonary macrophage transplantation therapy of hereditary PAP in mice.

Pulmonary macrophage transplantation (PMT) is a gene and cell transplantation approach in development as therapy for hereditary pulmonary alveolar proteinosis (hPAP), a surfactant accumulation disorder caused by mutations in CSF2RA/B (and murine homologs). We conducted a toxicology study of PMT of Csf2ra gene-corrected macrophages (mGM-Rα+Mϕs) or saline-control intervention in Csf2raKO or wild-type (WT) mice including single ascending dose and repeat ascending dose studies evaluating safety, tolerability, pharmacokinetics, and pharmacodynamics. Lentiviral-mediated Csf2ra cDNA transfer restored GM-CSF signaling in mGM-Rα+Mϕs. Following PMT, mGM-Rα+Mϕs engrafted, remained within the lungs, and did not undergo uncontrolled proliferation or result in bronchospasm, pulmonary function abnormalities, pulmonary or systemic inflammation, anti-transgene product antibodies, or pulmonary fibrosis. Aggressive male fighting caused a similarly low rate of serious adverse events in saline- and PMT-treated mice. Transient, minor pulmonary neutrophilia and exacerbation of pre-existing hPAP-related lymphocytosis were observed 14 days after PMT of the safety margin dose but not the target dose (5,000,000 or 500,000 mGM-Rα+Mϕs, respectively) and only in Csf2raKO mice but not in WT mice. PMT reduced lung disease severity in Csf2raKO mice. Results indicate PMT of mGM-Rα+Mϕs was safe, well tolerated, and therapeutically efficacious in Csf2raKO mice, and established a no adverse effect level and 10-fold safety margin.

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.

Slow cycling and durable Flt3+ progenitors contribute to hematopoiesis under native conditions.

The dynamics of the hematopoietic flux responsible for blood cell production in native conditions remains a matter of debate. Using CITE-seq analyses, we uncovered a distinct progenitor population that displays a cell cycle gene signature similar to the one found in quiescent hematopoietic stem cells. We further determined that the CD62L marker can be used to phenotypically enrich this population in the Flt3+ multipotent progenitor (MPP4) compartment. Functional in vitro and in vivo analyses validated the heterogeneity of the MPP4 compartment and established the quiescent/slow-cycling properties of the CD62L- MPP4 cells. Furthermore, studies under native conditions revealed a novel hierarchical organization of the MPP compartments in which quiescent/slow-cycling MPP4 cells sustain a prolonged hematopoietic activity at steady-state while giving rise to other lineage-biased MPP populations. Altogether, our data characterize a durable and productive quiescent/slow-cycling hematopoietic intermediary within the MPP4 compartment and highlight early paths of progenitor differentiation during unperturbed hematopoiesis.

The enhanced direct antiglobulin test in current practice has a limited impact on management of adult patients.

INTRODUCTION: The direct antiglobulin test (DAT) identifies immunoglobulin IgG and/or complement onthe red blood cell surface, allowing discrimination between immune and non-immunehemolysis. When the DAT is negative but there is clinical suspicion for immunehemolysis, an enhanced DAT can be sent to an immunohematology referencelaboratory (IRL). METHODOLOGY: This retrospective study assessed the volume of enhanced DATs at a large tertiarycare center and evaluated their impact on patient care. Enhanced DATs were sent on21 adult patients (January 2019 - January 2021) at the University of Pittsburgh MedicalCenter and Allegheny Health Network. Laboratory and clinical data were collected andanalyzed. RESULTS: Four out of 21 patients had positive tests (DAT and other serologic tests) at the localIRL. Enhanced DAT testing yielded positive results in an additional 5 patients butnegative or invalid results for 2 patients. High-dose steroid therapy was started in 12patients prior to receipt of enhanced DAT results. Enhanced DAT testing was sent amedian of 5 days after initiation of steroid therapy. For the patients trialed on steroids,the enhanced DAT results impacted medical decision-making in only 3 patients, and inonly one of those patients was the enhanced DAT positive despite a negative DAT at alocal IRL. In the non-steroid treated patients, enhanced DAT results did not contributeto clinical decision-making. CONCLUSION: Enhanced DATs generally did not impact medical decision-making in adults withhemolytic anemia.

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.

Third-Party and Patient-Specific Donor-Derived Virus-Specific T Cells Demonstrate Similar Efficacy and Safety for Management of Viral Infections after Hematopoietic Stem Cell Transplantation in Children and Young Adults.

Infections with double-stranded DNA viruses are a common complication after hematopoietic stem cell transplantation (HSCT) and cause significant morbidity and mortality in the post-transplantation period. Both donor-derived (DD) and third-party (TP) virus-specific T cells (VSTs) have shown efficacy and safety in viral management following HSCT in children and young adults. Owing to a greater degree of HLA matching between the recipient and stem cell donor, DD VSTs potentially persist longer in circulation compared to TP VSTs, because they are collected from a well-matched donor. However, TP VSTs are more easily accessible, particularly for smaller transplantation centers that do not have VST manufacturing capabilities, and more economical than creating a customized product for each transplant recipient. We conducted the present study to compare clinical efficacy and safety outcomes for DD VSTs and TP VSTs in a large cohort of pediatric and young adult HSCT recipients and to determine whether DD VSTs are associated with improved outcomes owing to potentially longer persistence in the recipient's circulation. This retrospective cohort study included 145 patients who received VSTs at Cincinnati Children's Hospital Medical Center (CCHMC) between 2017 and 2021 for the treatment of adenovirus, BK virus, cytomegalovirus, and/or Epstein-Barr virus. Viruses were detected using quantitative polymerase chain reaction. Patients received VSTs on a DD (NCT02048332) or TP (NCT02532452) protocol, and VST products for both protocols were manufactured in an identical fashion. The primary study outcome was clinical response to VSTs, evaluated 4 weeks after VST administration, defined as decrease in viral load to under the inclusion thresholds, or resolution of symptoms of invasive viral infection, without the need for additional conventional antiviral medication following VST administration. Secondary outcomes included graft-versus-host-disease, transplant-associated thrombotic microangiopathy, renal function, hospital length of stay, and overall survival at 30 days and 100 days after VST administration and 1 year after HSCT. Statistical analysis was performed using the Fisher exact test or chi-square test. An unpaired t test was used to compare continuous variables. The study group comprised 77 patients in the DD cohort and 68 patients in the TP cohort. Eighteen patients in the TP cohort underwent HSCT at CCHMC, and the other 50 underwent HSCT at other institutions and presented to CCHMC solely for VST administration. There was no statistically significant difference in clinical response rates between DD and TP cohorts (65.6% versus 62.7%; odds ratio [OR], 1.162; 95% confidence interval [CI], .619 to 2.164; P = .747). There were no significant differences in secondary outcomes between the 2 cohorts. The percentage of patients requiring multiple infusions for a clinical response did not differ significantly between the DD and TP cohorts (38.2% versus 32.5%; OR, .780; 95% CI, .345 to 1.805; P = .666). We found no significant difference in clinical response rate between DD VSTs and TP VSTs and a similar safety profile. Our data suggest that TP VSTs may be sufficient to control viral infection until immune reconstitution occurs despite the potential for more rapid VST clearance compared to DD VSTs. The lack of significant differences between DD VSTs and TP VSTs is an important finding, indicating that it is not necessary for every transplant center to manufacture customized DD VSTs, and that TP VSTs are a satisfactory substitute.

Rho GTPase Signaling in Platelet Regulation and Implication for Antiplatelet Therapies.

Platelets play a vital role in regulating hemostasis and thrombosis. Rho GTPases are well known as molecular switches that control various cellular functions via a balanced GTP-binding/GTP-hydrolysis cycle and signaling cascade through downstream effectors. In platelets, Rho GTPases function as critical regulators by mediating signal transduction that drives platelet activation and aggregation. Mostly by gene targeting and pharmacological inhibition approaches, Rho GTPase family members RhoA, Rac1, and Cdc42 have been shown to be indispensable in regulating the actin cytoskeleton dynamics in platelets, affecting platelet shape change, spreading, secretion, and aggregation, leading to thrombus formation. Additionally, studies of Rho GTPase function using platelets as a non-transformed model due to their anucleated nature have revealed valuable information on cell signaling principles. This review provides an updated summary of recent advances in Rho GTPase signaling in platelet regulation. We also highlight pharmacological approaches that effectively inhibited platelet activation to explore their possible development into future antiplatelet therapies.

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.

Mesenchymal stromal cells, metabolism, and mitochondrial transfer in bone marrow normal and malignant hematopoiesis.

Hematopoietic stem cell (HSC) transplantation-based treatments are in different phases of clinical development, ranging from current therapies to a promise in the repair and regeneration of diseased tissues and organs. Mesenchymal stromal/stem cells (MSCs), which are fibroblast-like heterogeneous progenitors with multilineage differentiation (osteogenic, chondrogenic, and adipogenic) and self-renewal potential, and exist in the bone marrow (BM), adipose, and synovium, among other tissues, represent one of the most widely used sources of stem cells in regenerative medicine. MSCs derived from bone marrow (BM-MSCs) exhibit a variety of traits, including the potential to drive HSC fate and anti-inflammatory and immunosuppressive capabilities via paracrine activities and interactions with the innate and adaptive immune systems. The role of BM-MSC-derived adipocytes is more controversial and may act as positive or negative regulators of benign or malignant hematopoiesis based on their anatomical location and functional crosstalk with surrounding cells in the BM microenvironment. This review highlights the most recent clinical and pre-clinical findings on how BM-MSCs interact with the surrounding HSCs, progenitors, and immune cells, and address some recent insights on the mechanisms that mediate MSCs and adipocyte metabolic control through a metabolic crosstalk between BM microenvironment cells and intercellular mitochondrial transfer in normal and malignant hematopoiesis.

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.

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.

A Limited Role for AMD3100 Induced Stem Cell Mobilization for Modulation of Thoracic Trauma Outcome.

ABSTRACT: Thoracic trauma is a major cause of mortality due to the associated inflammatory acute respiratory distress syndrome and morbidity due to impaired tissue regeneration. Trauma-induced lung inflammation is characterized by the early recruitment of cells with pro- or anti-inflammatory activity to the lung. Therapeutic interventions reducing the level of tissue inflammation may result in decreased tissue damage and improved healing and recovery. Stem cells might be able to improve trauma outcome via immunomodulation or by enhancing tissue regeneration.Here, we describe the migratory dynamics of murine mesenchymal, hematopoietic and endothelial stem and progenitor cells (SPCs) as well as mature inflammatory cells (monocytes, neutrophils, lymphocytes) to peripheral blood (PB) and lung tissue between 0.2 and 48 h post-blunt chest trauma (TXT). We demonstrate that the kinetics of immune cell and SPC distribution upon trauma are both cell-type and tissue-dependent. We identified a transient, early increase in the number of inflammatory cells in PB and lung at 2 h post-TXT and a second wave of infiltrating SPCs in lungs by 48 h after TXT induction, suggesting a role for SPCs in tissue remodeling after the initial inflammatory phase. Cxcl12/Cxcr4 blockade by AMD3100 within the first 6 h after TXT, while inducing a strong and coordinated mobilization of SPCs and leukocytes to PB and lung tissue, did not significantly affect TXT associated inflammation or tissue damage as determined by inflammatory cytokine levels, plasma markers for organ function, lung cell proliferation and survival, and myofibroblast/fibroblast ratio in the lung. Further understanding the dynamics of the distribution of endogenous SPCs and inflammatory cells will therefore be indispensable for stem cell-based or immunomodulation therapies in trauma.

P2RY14 cAMP signaling regulates Schwann cell precursor self-renewal, proliferation, and nerve tumor initiation in a mouse model of neurofibromatosis.

Neurofibromatosis type 1 (NF1) is characterized by nerve tumors called neurofibromas, in which Schwann cells (SCs) show deregulated RAS signaling. NF1 is also implicated in regulation of cAMP. We identified the G-protein-coupled receptor (GPCR) P2ry14 in human neurofibromas, neurofibroma-derived SC precursors (SCPs), mature SCs, and mouse SCPs. Mouse Nf1-/- SCP self-renewal was reduced by genetic or pharmacological inhibition of P2ry14. In a mouse model of NF1, genetic deletion of P2ry14 rescued low cAMP signaling, increased mouse survival, delayed neurofibroma initiation, and improved SC Remak bundles. P2ry14 signals via Gi to increase intracellular cAMP, implicating P2ry14 as a key upstream regulator of cAMP. We found that elevation of cAMP by either blocking the degradation of cAMP or by using a P2ry14 inhibitor diminished NF1-/- SCP self-renewal in vitro and neurofibroma SC proliferation in in vivo. These studies identify P2ry14 as a critical regulator of SCP self-renewal, SC proliferation, and neurofibroma initiation.

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.

Scheduled administration of virus-specific T cells for viral prophylaxis after pediatric allogeneic stem cell transplant.

Infections with double-stranded DNA viruses are a significant cause of morbidity and mortality in pediatric patients following allogeneic hematopoietic stem cell transplantation (HSCT). Virus-specific T-cell therapies (VSTs) have been shown to be an effective treatment for infections with adenovirus, BK virus, cytomegalovirus (CMV), and Epstein-Barr virus (EBV). To date, prophylactic regimens to prevent or mitigate these infections using conventional antiviral medications provide suboptimal response rates. Here we report on a clinical trial (NCT03883906) performed to assess the feasibility of rapid manufacturing and early infusion of quadrivalent VSTs generated from stem cell donors ("donor-derived VSTs") into allogeneic HSCT recipients with minimal or absent viremia. Patients were eligible to receive scheduled VSTs as early as 21 days after stem cell infusion. Twenty-three patients received scheduled VSTs. Twenty of 23 patients had no viremia at the time of infusion, while 3 patients had very low-level BK viremia. Two developed clinically significant graft-versus-host disease (GVHD), although this incidence was not outside of expected incidence early after HSCT, and both were successfully treated with systemic corticosteroids (n = 2). Five patients were deemed treatment failures. Three developed subsequent significant viremia/viral disease (n = 3). Eighteen patients did not fail treatment, 7 of whom did not develop any viremia, while 11 developed low-level, self-limited viremia that resolved without further intervention. No infusion reactions occurred. In conclusion, scheduled VSTs appear to be safe and potentially effective at limiting serious complications from viral infections after allogeneic transplantation. A randomized study comparing this scheduled approach to the use of VSTs to treat active viremia is ongoing.