Hypoxia Controls the Glycome Signature and Galectin-8-Ligand Axis to Promote Protumorigenic Properties of Metastatic Melanoma.

The prognosis for patients with metastatic melanoma (MM) involving distant organs is grim, and treatment resistance is potentiated by tumor-initiating cells (TICs) that thrive under hypoxia. MM cells, including TICs, express a unique glycome featuring i-linear poly-N-acetyllactosamines through the loss of I-branching enzyme, ß1,6 N-acetylglucosaminyltransferase 2. Whether hypoxia instructs MM TIC development by modulating the glycome signature remains unknown. In this study, we explored hypoxia-dependent alterations in MM glycome‒associated genes and found that ß1,6 N-acetylglucosaminyltransferase 2 was downregulated and a galectin (Gal)-8-ligand axis, involving both extracellular and cell-intrinsic Gal-8, was induced. Low ß1,6 N-acetylglucosaminyltransferase 2 levels correlated with poor patient outcomes, and patient serum samples were elevated for Gal-8. Depressed ß1,6 N-acetylglucosaminyltransferase 2 in MM cells upregulated TIC marker, NGFR/CD271, whereas loss of MM cell‒intrinsic Gal-8 markedly lowered NGFR and reduced TIC activity in vivo. Extracellular Gal-8 bound preferentially to i-linear poly-N-acetyllactosamines on N-glycans of the TIC marker and prometastatic molecule CD44, among other receptors, and activated prosurvival factor protein kinase B. This study reveals the importance of hypoxia governing the MM glycome by enforcing i-linear poly-N-acetyllactosamine and Gal-8 expression. This mechanistic investigation also uncovers glycome-dependent regulation of pro-MM factor, NGFR, implicating i-linear poly-N-acetyllactosamine and Gal-8 as biomarkers and therapeutic targets of MM.

International Society of Blood Transfusion Working Party on Red Cell Immunogenetics and Blood Group Terminology Report of Basel and three virtual business meetings: Update on blood group systems.

BACKGROUND AND OBJECTIVES: Under the ISBT, the Working Party (WP) for Red Cell Immunogenetics and Blood Group Terminology is charged with ratifying blood group systems, antigens and alleles. This report presents the outcomes from four WP business meetings, one located in Basel in 2019 and three held as virtual meetings during the COVID-19 pandemic in 2020 and 2021. MATERIALS AND METHODS: As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. New blood group systems and antigens were approved and named according to the serologic, genetic, biochemical and cell biological evidence presented. RESULTS: Seven new blood group systems, KANNO (defined numerically as ISBT 037), SID (038), CTL2 (039), PEL (040), MAM (041), EMM (042) and ABCC1 (043) were ratified. Two (039 and 043) were de novo discoveries, and the remainder comprised reported antigens where the causal genes were previously unknown. A further 15 blood group antigens were added to the existing blood group systems: MNS (002), RH (004), LU (005), DI (010), SC (013), GE (020), KN (022), JMH (026) and RHAG (030). CONCLUSION: The ISBT now recognizes 378 antigens, of which 345 are clustered within 43 blood group systems while 33 still have an unknown genetic basis. The ongoing discovery of new blood group systems and antigens underscores the diverse and complex biology of the red cell membrane. The WP continues to update the blood group antigen tables and the allele nomenclature tables. These can be found on the ISBT website (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/).

Production and nonclinical evaluation of an autologous iPSC-derived platelet product for the iPLAT1 clinical trial.

Donor-derived platelets are used to treat or prevent hemorrhage in patients with thrombocytopenia. However, ∼5% or more of these patients are complicated with alloimmune platelet transfusion refractoriness (allo-PTR) due to alloantibodies against HLA-I or human platelet antigens (HPA). In these cases, platelets from compatible donors are necessary, but it is difficult to find such donors for patients with rare HLA-I or HPA. To produce platelet products for patients with aplastic anemia with allo-PTR due to rare HPA-1 mismatch in Japan, we developed an ex vivo good manufacturing process (GMP)-based production system for an induced pluripotent stem cell-derived platelet product (iPSC-PLTs). Immortalized megakaryocyte progenitor cell lines (imMKCLs) were established from patient iPSCs, and a competent imMKCL clone was selected for the master cell bank (MCB) and confirmed for safety, including negativity of pathogens. From this MCB, iPSC-PLTs were produced using turbulent flow bioreactors and new drugs. In extensive nonclinical studies, iPSC-PLTs were confirmed for quality, safety, and efficacy, including hemostasis in a rabbit model. This report presents a complete system for the GMP-based production of iPSC-PLTs and the required nonclinical studies and thus supports the iPLAT1 study, the first-in-human clinical trial of iPSC-PLTs in a patient with allo-PTR and no compatible donor using the autologous product. It also serves as a comprehensive reference for the development of widely applicable allogeneic iPSC-PLTs and other cell products that use iPSC-derived progenitor cells as MCB.

Dual preparation of plasma and platelet concentrates in platelet additive solution from platelet concentrates in plasma using a novel filtration system.

BACKGROUND AND OBJECTIVES: Platelet concentrates suspended in a platelet additive solution (PAS-PC) are associated with a reduction in allergic response and are suitable for preparing pathogen-inactivated PC. We aimed to develop an efficient platform for the dual preparation of PAS-PC and platelet-poor plasma. MATERIALS AND METHODS: PAS-PC was prepared in six steps by using a hollow-fibre system based on cross-flow filtration: priming, loading PC, loading PAS, collection of filtered liquid (flow-through) and collection of platelets by washing with PAS followed by washing with air. In this study, the efficacy of platelet and plasma protein recovery and characteristics of recovered PAS-PC and flow-through plasma were analysed in detail. RESULTS: Recoveries of platelet in PAS-PC and plasma protein in the flow-through were 95.4% ± 3.7% and 61.6% ± 5.0%, respectively. The residual plasma protein in PAS-PC was 34.1% ± 2.8%. Although the expression level of CD62P, a platelet activation marker, in recovered platelets was approximately 1.2-fold of that in original platelets, swirling patterns were well retained, and aggregation in PAS-PC was not visible. Agonist-induced aggregabilities, platelet morphology and hypotonic shock recovery were conserved. The patterns of plasma protein and lipoprotein in the flow-through were comparable with those in the original PCs. The multimeric pattern analysis of VWF remained unaltered. CONCLUSION: We propose a highly efficient preparation system that enables the simultaneous production of PAS-PC and platelet-poor plasma. It also achieves a high recovery of functionally well-retained platelets with very low activation.

Erythrocyte CD55 mediates the internalization of Plasmodium falciparum parasites.

Invasion of human erythrocytes by the malaria parasite Plasmodium falciparum is a multi-step process. Previously, a forward genetic screen for P. falciparum host factors identified erythrocyte CD55 as essential for invasion, but its specific role and how it interfaces with the other factors that mediate this complex process are unknown. Using CRISPR-Cas9 editing, antibody-based inhibition, and live cell imaging, here we show that CD55 is specifically required for parasite internalization. Pre-invasion kinetics, erythrocyte deformability, and echinocytosis were not influenced by CD55, but entry was inhibited when CD55 was blocked or absent. Visualization of parasites attached to CD55-null erythrocytes points to a role for CD55 in stability and/or progression of the moving junction. Our findings demonstrate that CD55 acts after discharge of the parasite's rhoptry organelles, and plays a unique role relative to all other invasion receptors. As the requirement for CD55 is strain-transcendent, these results suggest that CD55 or its interacting partners may hold potential as therapeutic targets for malaria.

UV light-emitting diode (UV-LED) at 265 nm as a potential light source for disinfecting human platelet concentrates.

The risk of sepsis through bacterial transmission is one of the most serious problems in platelet transfusion. In processing platelet concentrates (PCs), several methods have been put into practice to minimize the risk of bacterial transmission, such as stringent monitoring by cultivation assays and inactivation treatment by photoirradiation with or without chemical agents. As another potential option, we applied a light-emitting diode (LED) with a peak emission wavelength of 265 nm, which has been shown to be effective for water, to disinfect PCs. In a bench-scale UV-LED exposure setup, a 10-min irradiation, corresponding to an average fluence of 9.2 mJ/cm2, resulted in >2.0 log, 1.0 log, and 0.6 log inactivation (mean, n = 6) of Escherichia coli, Staphylococcus aureus, and Bacillus cereus, respectively, in non-diluted plasma PCs. After a 30-min exposure, platelet counts decreased slightly (18 ± 7%: mean ± SD, n = 7); however, platelet surface expressions of CD42b, CD61, CD62P, and PAC-1 binding did not change significantly (P>0.005), and agonist-induced aggregation and adhesion/aggregation under flow conditions were well maintained. Our findings indicated that the 265 nm UV-LED has high potential as a novel disinfection method to ensure the microbial safety of platelet transfusion.

Frequency of allotype "b" in human platelet antigen 1 to 29 systems among blood donors in Japan estimated using high-resolution melt analysis.

BACKGROUND: Antibodies against human platelet antigens (HPAs) cause thrombocytopenias. It is thus important to know the frequency of "b" allotypes in each HPA system for the diagnosis and treatment of anti-HPA antibody-mediated thrombocytopenia. STUDY DESIGN AND METHODS: Genomic DNA was extracted from peripheral blood cells obtained from 2170 blood donors in Japan and was subjected to high-resolution melt (HRM) analysis using polymerase chain reaction for each of the HPA genes, using 23 primer pairs. For genotyping, the resulting amplicons were classified based on their HRM curves. In some cases, direct sequence analysis was performed after HRM analysis to determine nucleotide substitutions. In cases where amino acid substitutions were predicted, protein expression levels were examined in a cell line using 293T cells. RESULTS: The frequencies of each of the HPA-b genotypes were as follows: HPA-1b, 0.4%; HPA-2b, 11.8%; HPA-3b, 41.3%; HPA-4b, 0.8%; HPA-5b, 4.3%; HPA-6b, 1.9%; HPA-15b, 48.8%; HPA-21b, 0.6%; and "b" allotype in the other HPA systems, 0.0%. Twenty-eight variants were found; nine of them were predicted to cause amino acid substitution. However, expression analysis revealed that they did not affect protein expression levels on the cell surface. CONCLUSION: Nine HPA systems are of primary importance in Japan in potentially triggering thrombocytopenia via the HPA antibodies. Similar studies in other countries or races, together with ours, could provide basic information for clinicians in multiethnic societies.

The Kg-antigen, RhAG with a Lys164Gln mutation, gives rise to haemolytic disease of the newborn.

The Kg-antigen was first discovered in an investigation of a mother whose infant had haemolytic disease of the newborn (HDN). The antibody against the Kg-antigen is believed to be responsible for HDN. The Kg-antigen is provisionally registered under the number 700045, according to the Red Cell Immunogenetics and Blood Group Terminology. However, the molecular nature of the Kg-antigen has remained a mystery for over 30 years. In this study, a monoclonal antibody against the Kg-antigen and the recombinant protein were developed that allowed for the immunoprecipitation analysis. Immunoprecipitants from the propositus' red blood cell ghosts were subjected to mass spectrometry analysis, and DNA sequence analysis of the genes was also performed. A candidate for the Kg-antigen was molecularly isolated and confirmed to be a determinant of the Kg-antigen by cell transfection and flow cytometry analyses. The Kg-antigen and the genetic mutation were then screened for in a Japanese population. The molecular nature of the Kg-antigen was shown to be RhAG with a Lys164Gln mutation. Kg phenotyping further clarified that 0.22% of the Japanese population studied was positive for the Kg-antigen. These findings provide important information on the Kg-antigen, which has been clinically presumed to give rise to HDN.

A more efficient preparation system for HLA-eliminated platelets.

BACKGROUND AND OBJECTIVES: Although HLA-eliminated platelets can facilitate transfusions to patients possessing HLA antibodies, no such products are currently available commercially perhaps because the platelet collection rate is not yet economically viable. We have improved this process' efficiency by employing a hollow-fibre system at the last step of the production process after an acid and a reaction buffer have been washed out conventionally by centrifugation. MATERIALS AND METHODS: HLA-eliminated platelets were prepared via four distinct steps: chilled on ice, treated with an acid solution, diluted and finally washed using the hollow-fibre system. The efficiency of this platelet recovery process was determined. The resulting products' platelet characteristics, including a capacity for HLA expression, were evaluated in vitro and compared in detail to their corresponding originals. RESULTS: The average efficiency of platelet recovery was 91%. Although the expression levels of CD62P, a molecular marker for platelet activation, were approximately threefold higher on new platelets than on the original platelets, their HLA expression levels were lower. The phagocytosis assay, with monoclonal antibodies and cognate HLA antibody-containing sera, suggested that HLA-ABC molecules on the cell surface were sufficiently removed. The platelet functions, including the agonist-induced aggregability and adherence/aggregability of the collagen-coated plates under certain conditions, were conserved and not significantly different from the original ones. CONCLUSION: We propose a novel preparation system for producing HLA-eliminated platelets without centrifugation, which ensures a highly efficient, and therefore, much more economical method of platelet recovery that also retains their key functionality.

International Society of Blood Transfusion Working Party on Red Cell Immunogenetics and Blood Group Terminology: Report of the Dubai, Copenhagen and Toronto meetings.

BACKGROUND AND OBJECTIVES: The International Society of Blood Transfusion (ISBT) Working Party for Red Cell Immunogenetics and Blood Group Terminology meets in association with the ISBT congress and has met three times since the last report: at the international meetings held in Dubai, United Arab Emirates, September 2016 and Toronto, Canada, June 2018; and at a regional congress in Copenhagen, Denmark, June 2017 for an interim session. METHODS: As in previous meetings, matters pertaining to blood group antigen nomenclature and classification were discussed. New blood group antigens were approved and named according to the serologic and molecular evidence presented. RESULTS AND CONCLUSIONS: Fifteen new blood group antigens were added to eight blood group systems. One antigen was made obsolete based on additional data. Consequently, the current total of blood group antigens recognized by the ISBT is 360, of which 322 are clustered within 36 blood groups systems. The remaining 38 antigens are currently unassigned to a known system. Clinically significant blood group antigens continue to be discovered, through serology/sequencing and/or recombinant or genomic technologies.

Erythrocytes lacking the Langereis blood group protein ABCB6 are resistant to the malaria parasite Plasmodium falciparum.

The ATP-binding cassette transporter ABCB6 was recently discovered to encode the Langereis (Lan) blood group antigen. Lan null individuals are asymptomatic, and the function of ABCB6 in mature erythrocytes is not understood. Here, we assessed ABCB6 as a host factor for Plasmodium falciparum malaria parasites during erythrocyte invasion. We show that Lan null erythrocytes are highly resistant to invasion by P. falciparum, in a strain-transcendent manner. Although both Lan null and Jr(a-) erythrocytes harbor excess porphyrin, only Lan null erythrocytes exhibit a P. falciparum invasion defect. Further, the zoonotic parasite P. knowlesi invades Lan null and control cells with similar efficiency, suggesting that ABCB6 may mediate P. falciparum invasion through species-specific molecular interactions. Using tandem mass tag-based proteomics, we find that the only consistent difference in membrane proteins between Lan null and control cells is absence of ABCB6. Our results demonstrate that a newly identified naturally occurring blood group variant is associated with resistance to Plasmodium falciparum.

Loss of GCNT2/I-branched glycans enhances melanoma growth and survival.

Cancer cells often display altered cell-surface glycans compared to their nontransformed counterparts. However, functional contributions of glycans to cancer initiation and progression remain poorly understood. Here, from expression-based analyses across cancer lineages, we found that melanomas exhibit significant transcriptional changes in glycosylation-related genes. This gene signature revealed that, compared to normal melanocytes, melanomas downregulate I-branching glycosyltransferase, GCNT2, leading to a loss of cell-surface I-branched glycans. We found that GCNT2 inversely correlated with clinical progression and that loss of GCNT2 increased melanoma xenograft growth, promoted colony formation, and enhanced cell survival. Conversely, overexpression of GCNT2 decreased melanoma xenograft growth, inhibited colony formation, and increased cell death. More focused analyses revealed reduced signaling responses of two representative glycoprotein families modified by GCNT2, insulin-like growth factor receptor and integrins. Overall, these studies reveal how subtle changes in glycan structure can regulate several malignancy-associated pathways and alter melanoma signaling, growth, and survival.

Pulmonary Involvement of Acute Myeloid Leukemia Mimicking Transfusion-related Acute Lung Injury.

Transfusion-related acute lung injury (TRALI) is defined as a new episode of acute lung injury (ALI) occurring during transfusion or within 6 hours of transfusion completion. A 66-year-old man suffering from acute myeloid leukemia developed acute respiratory distress syndrome after platelet transfusion. TRALI was diagnosed clinically, but an autopsy showed leukemic cells in diffuse pulmonary edema. Anti-human neutrophil antigen (HNA)-3a antibodies were detected in the donor serum, and the HNA-3 genotype of the patient was identified as a/a. This case was considered to represent pulmonary involvement of acute myeloid leukemia, rather than TRALI. A revision of the definition of TRALI accounting for hematological malignancies should therefore be considered.

Mitochondrial damage-associated molecular patterns as potential proinflammatory mediators in post-platelet transfusion adverse effects.

BACKGROUND: Platelet concentrates (PCs) are the most common blood components eliciting nonhemolytic transfusion reactions (NHTRs), such as allergic transfusion reactions and febrile reactions. However, the precise mechanisms of NHTRs in PC transfusion remain largely unknown. Previous studies reported that mitochondria-derived damage-associated molecular patterns (DAMPs) could be important mediators of innate cell inflammation. Platelets (PLTs) represent a major reservoir of mitochondria in the blood circulation. The aim of this study was to determine the possible involvement of mitochondrial DAMPs in NHTRs. STUDY DESIGN AND METHODS: The amount of mitochondrial DAMPs was determined as an index of total copy numbers of mitochondrial DNA (mtDNA), including mtDNA itself and free mitochondria, using quantitative real-time polymerase chain reaction. To examine whether neutrophils, monocytes, and basophils were activated by mitochondrial DAMPs in vitro, an in vitro whole blood cell culture assay was performed. RESULTS: In blood components associated with NHTRs, the mean total mtDNA concentration was highest in PCs followed in order by fresh-frozen plasma and red blood cells. The amount of mtDNA in NHTR PCs was higher than that in control PCs without NHTRs. The mitochondrial DAMPs present in NHTR PCs was high enough to activate neutrophils, monocytes, and basophils, when costimulated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine or HLA antibodies. CONCLUSION: PLT-derived mitochondrial DAMPs are candidate risk factors for the onset of NHTRs.

Expression levels of ABCG2 on cord red blood cells and study of fetal anemia associated with anti-Jr(a).

BACKGROUND: The Jr(a) antigen of JR blood group systems is located on ABCG2 and Jr(a-) subjects whose red blood cells (RBCs) lack ABCG2 have been identified mostly among the Japanese. Although anti-Jr(a) can cause fetal anemia, little is known regarding its mechanism. STUDY DESIGN AND METHODS: We reviewed clinical courses of all reported cases with fetal anemia due to anti-Jr(a) . We analyzed the ABCG2 expressions of cord RBCs at various gestational ages. We examined the effects of sera containing anti-Jr(a) from three pregnancies with fetal anemia or monoclonal anti-Jr(a) on erythropoiesis and phagocytosis. We also examined epitopes of anti-Jr(a) . RESULTS: Case series suggested that the majority of fetal anemia with anti-Jr(a) may not be progressive in the later gestational ages. ABCG2 expression levels of cord RBCs were significantly higher than those of adults and neonates with high individual variation and gradually decreased with advancing gestational ages. Anti-Jr(a) did not significantly impact erythroid colony formation, although we detected a tendency toward the suppression of erythroid burst-forming unit formation by anti-Jr(a) using feline marrow cells. Anti-Jr(a) did not induce phagocytosis of sensitized RBCs by monocytes. While many anti-Jr(a) recognized the same regions as a monoclonal anti-ABCG2, 5D3, epitopes of anti-Jr(a) did not correlate with the incidence of fetal anemia. CONCLUSION: ABCG2 expression levels in cord RBCs are higher than those of adults, and the change of ABCG2 expression in erythroid lineage cells may influence the clinical course of fetal anemia with anti-Jr(a) , although we could not detect significant effects of anti-Jr(a) on erythroid colony formation or phagocytosis.

High-resolution melting analysis as an alternative method for human neutrophil antigen genotyping.

Human neutrophil antigen (HNA)-typed granulocyte panels are widely used to screen for the presence of HNA antibodies and to determine antibody specificity. Many laboratories screen donors for HNA genotypes using low-throughput methods such as allele-specific polymerase chain reaction (PCR), PCR-restriction fragment-length polymorphism, and multiplex PCR. In the present study, we used a high-resolution melting (HRM) analysis to determine HNA genotypes. For the HRM analysis, purified genomic DNA samples were amplified via PCR with HNA-specific primers. Nucleotide substitutions in genes encoding HNAs were differentiated on the basis of the HRM curves, and the results of HRM and DNA sequencing analyses were determined to be in complete agreement. The gene frequency of HNA-1a, -1b, -1c, -3a, -3b, -4a, -4b, -5a, and -5b in the Japanese population was consistent with the previous reports. Our results suggest that HRM analysis can be used for genotyping HNA antigens determined by single nucleotide substitutions.