Glycophorin Research Articles

A Pharmacologic Approach Using Pomalidomide and Dexamethasone to Study Stage-Specific Regulation of Erythroid Progenitors

The erythroid progenitors, burst forming unit-erythroid (BFU-E) and colony forming unit-erythroid (CFU-E), control red cell production through the integration of a variety of self-renewal and differentiation signals. The identification of these signals and their effects on erythroid progenitor populations are of therapeutic interest for human disorders characterized by decreased red cell production such as Diamond Blackfan Anemia (DBA). We have previously documented that pomalidomide acts at the BFU-E stage and dexamethasone at the CFU-E stage in in vitro models of human erythropoiesis. Therefore, these two agents represent potential pharmacologic interventions for the treatment of human diseases with disordered erythropoiesis. Here, we studied the mechanisms by which pomalidomide and dexamethasone enhance erythroid progenitor numbers in cord blood and adult bone marrow-derived CD34+ hematopoietic stem and progenitor cells (HSPC).Pomalidomide increased adult and fetal BFU-E numbers in both serum-free and serum-containing in vitro erythroid culture systems. CFU-E numbers and terminal erythroblasts were also transiently attenuated suggesting that pomalidomide mediates increased BFU-E in part through delayed progression to erythropoietin-sensitive progenitors and precursor stages. Transforming growth factor-β (TGF-β) has previously been shown to regulate the transition from BFU-E to CFU-E, and therefore, we investigated whether pomalidomide modulates TGF-β signaling. We noted accelerated differentiation as assessed by glycophorin A (GPA) expression of CD34+ HSPCs treated with TGF-β1, and the addition of pomalidomide completely abrogated TGF-β1 mediated differentiation. This effect was not due to inhibition of TGF-β1 receptors or kinases as treatment of NIH 3T3 cells with TGF-β1 ± pomalidomide failed to prevent SMAD2 phosphorylation. Only CD34+ HSPCs differentiated in the presence of pomalidomide exhibited reduced sensitivity to TGF-β1. These results suggest that pomalidomide alters erythroid and/or earlier hematopoietic progenitor differentiation to enrich a population of TGF-β1 insensitive cells and/or affects the activity of downstream TGF-β1 effectors.Dexamethasone, in contrast to pomalidomide, was found to specifically increase the number of CFU-E progenitors. Elevated CFU-E self-renewal was associated with an accumulation of cells in G0/G1 of the cell cycle, which suggests that alterations to cell cycle progression may contribute to the regulation of self-renewal genes in CFU-E. In contrast to adult-derived CFU-E, dexamethasone failed to affect CFU-E numbers or cell cycle status in cord blood derived CFU-Es. We investigated the expression of glucocorticoid receptor isoforms (GRα and GRβ) in erythroid progenitors, which revealed that BFU-E and CFU-E differentially express GR isoforms. BFU-E expressed comparable levels of GRα and the hypothesized dominant-negative GRβ, where as only GRα transcript was measured in CFU-E. These expression patterns were similar between adult and cord blood-derived progenitors. Despite similar GR levels, adult-derived erythroid progenitors, but not cord blood derived progenitors, were shown to translocate GRα to the nucleus after a 24hr pulse of dexamethasone suggesting that post-translational mechanisms may govern dexamethasone response in erythroid progenitors.These findings strongly indicate that pharmacologic modulation of the TGF-β and glucocorticoid-signaling pathways can effectively stimulate erythroid progenitor self-renewal. We are currently elucidating specific targets for pomalidomide and dexamethasone in BFU-E and CFU-E, respectively, through proteomics and transcriptome approaches, and investigating the efficacy of mono- and combination therapies using peripheral blood-derived CD34+ HSPCs from DBA patients to study their therapeutic efficacy. DisclosuresNo relevant conflicts of interest to declare.

Diagnostic value of glycophorin-A in comparison with P57 immunohistochemical staining method in differentiating complete and partial molar pregnancies

IntroductionCurrent histomorphological criteria in distinguishing two subtypes of hydatidiform moles has considerable inter-observer variability and limitations. In this regard, ancillary studies can aid pathologist to obtain an accurate diagnosis. Herein, we evaluated the utility of Glycophorin-A (GLA) in differentiating complete and partial moles. Materials and methodsIn this case-control study, formalin-fixed paraffin-embedded blocks of 47 patients with pathologic diagnosis of complete and 42 partial hydatidiform moles were included and the diagnoses were confirmed by immunohistochemistry (IHC) for P57. Sections from all samples were stained for GLA using IHC method. Using 2 × 2 tables, the sensitivity, specifity, Positive and Negative Predictive Values (PPV and NPV) as well as accuracy of GLA were determined. ResultsPrimary pathologic diagnosis was changed in 7.1% and types of hydatidiform mole were specified in 11.9% of the cases after review of the slides and IHC study for P57. NRBCs were found in 52.7% of the PM cases and none of CMs by pathologist in H&E sections. IHC study for GLA revealed positive result in one case of complete moles (2%) and 31 case of partial mole samples (73.8%). It was negative in 98% of the complete mole and 11 (26.2%) of partial mole cases. DiscussionThe results of this study showed a significant association between GLA immunoreactivity and type of molar pregnancy. Diagnostic sensitivity, specificity and accuracy of this marker for discrimination of molar pregnancy were 73.8%, 98% and 86.5%, respectively. Therefore, this marker can be utilized in differentiating partial and complete hydatidiform mole.

In silico molecular dynamics of human glycophorin A (GPA) extracellular structure

Background: Glycophorin A (GPA) is one of two glycoproteins found on the surface of human red blood cells (RBCs) that constitute the MNS blood group system. The structure of GPA’s extracellular domain is unknown despite previous attempts using X-ray crystallography and NMR spectroscopy. As a result, there is a knowledge gap regarding antigen presentation. This hinders the production of monoclonal antibodies (mAbs) against MNS antigens.

Near-Infrared Autofluorescence in Atherosclerosis Associates With Ceroid and Is Generated by Oxidized Lipid-Induced Oxidative Stress.

[Figure: see text].

Determination of the Bacteriostatic Activity of Glycophorin Preparations from Red Blood Cell (RBC) Membranes of Yellow Tail and Red Sea Bream

Glycophorins (GPs) in red blood cell (RBC) membranes of carp (Cyprinus carpio L.) exhibit bacteriostatic activity against various gram-negative and gram-positive bacteria. To clarify the physiological activity of teleost fish GPs other than those from carp, we performed a sensitivity test using GP preparations from the RBC membranes of yellow tail and red sea bream. The sensitivity test for the growth of Micrococcus luteus was performed using the disc-plate method. These results suggested that the yellow tail and red sea bream GPs exhibit antibiotic activities.

The interaction between Glycophorin A (GPA) and Band 3 in the formation of the Wright b (Wrb ) antigen.

The interaction between Glycophorin A (GPA) and Band 3 in the formation of the Wright b (Wrb ) antigen.

Hotspots in Plasmodium and RBC Receptor-Ligand Interactions: Key Pieces for Inhibiting Malarial Parasite Invasion.

Protein-protein interactions (IPP) play an essential role in practically all biological processes, including those related to microorganism invasion of their host cells. It has been found that a broad repertoire of receptor-ligand interactions takes place in the binding interphase with host cells in malaria, these being vital interactions for successful parasite invasion. Several trials have been conducted for elucidating the molecular interface of interactions between some Plasmodium falciparum and Plasmodium vivax antigens with receptors on erythrocytes and/or reticulocytes. Structural information concerning these complexes is available; however, deeper analysis is required for correlating structural, functional (binding, invasion, and inhibition), and polymorphism data for elucidating new interaction hotspots to which malaria control methods can be directed. This review describes and discusses recent structural and functional details regarding three relevant interactions during erythrocyte invasion: Duffy-binding protein 1 (DBP1)–Duffy antigen receptor for chemokines (DARC); reticulocyte-binding protein homolog 5 (PfRh5)-basigin, and erythrocyte binding antigen 175 (EBA175)-glycophorin A (GPA).

Non-synonymous amino acid alterations in PfEBA-175 modulate the merozoite ligand's ability to interact with host's Glycophorin A receptor

The pathological outcome of malaria due to Plasmodium falciparum infection depends largely on erythrocyte invasion by blood-stage merozoites which employ a cascade of interactions occurring between parasite ligands and RBC receptors. In a previous study exploring the genetic diversity of region-II of PfEBA-175, a ligand that plays a crucial part in parasite's RBC entry through Glycophorin A (GPA) receptor, we demonstrated that F2 domain of region-II underwent positive selection in Indian P. falciparum population through the accumulation of non-synonymous polymorphisms. Here, we examine the functional impact of two highly prevalent non-synonymous alterations in F2, namely Q584E & E592A, using a battery of molecular, biophysical and in-silico techniques. Application of circular dichroism, FTIR, fluorescence spectroscopy reveals that secondary and three-dimensional folding of recombinant-F2 protein carrying 584E and 592A residues (F2-Mut) differs significantly from that carrying 584Q and 592E (F2-3D7). A comparison of spectroscopic and thermodynamic parameters shows that F2-Mut is capable of forming a complex with GPA with higher efficiency compared to F2-3D7. In silico docking predicts both artemisinin and artesunate possess the capacity of slipping into the GPA binding crevices of PfEBA-175 and disrupt PfEBA-GPA association. However, the estimated affinity of artesunate towards PfEBA-175 with 584E and 592A residues is higher than that of artemisinin. Thermodynamic parameters computed using isotherms are concordant with this in-silico prediction. Together, our data suggest that the presence of amino acid alterations in F2 provide structural and functional stability favoring PfEBA-GPA interaction and artesunate can efficiently disrupt the interaction between GPA and PfEBA-175 even carrying altered amino acid residues. The present study alerts the malaria research community by presenting evidence that the parasite is gaining evolutionary fitness by cultivating genetic alterations in many of its proteins.

Decreased erythrocyte binding of Siglec-9 increases neutrophil activation in sickle cell disease

Oxidative stress and inflammation promote vaso-occlusion in sickle cell disease (SCD). CD33-related Sialic acid-binding immunoglobulin-type lectins (CD33rSiglecs) are cell surface proteins that recognize sialic acids inhibit innate immune cell functions. We have shown that Siglec-9 on human neutrophils interact with erythrocyte sialic acids (prominently glycophorin-A (GYPA) to suppress neutrophil reactive oxygen species (ROS). We hypothesized that altered sickle erythrocyte membrane sialic acid leads to decreased Siglec-9 binding capability, and thus a decreased neutrophil oxidative burst. SS erythrocytes express significantly more sialic acid than AA erythrocytes (p = 0.02). SS erythrocytes displayed significantly less Siglec-9-Fc binding 39% ± 11 (mean ± SEM) compared to AA erythrocytes 78% ± 5 (p = 0.009). Treatment of AA erythrocytes with sialidase to remove sialic acid decreased binding to 3% ± 7.9 (p ≤ 0.001). When freshly isolated neutrophils were incubated with AA erythrocytes, neutrophils achieved 16% ± 6 of the oxidative burst exhibited by a stimulated neutrophil without erythrocytes. In contrast, neutrophils incubated with SS erythrocytes achieved 47% ± 6 of the oxidative burst (AA versus SS, p = 0.03). Stimulated neutrophils incubated with AA erythrocytes showed minimal NET formation while with SS erythrocytes NETs increased. SS erythrocytes are deficient in binding to neutrophil Siglec-9 which may contribute to the increased oxidative stress in SCD.

A GPI-Anchored Protein, CD109, Protects Hematopoietic Progenitor Cells from Erythroid Differentiation Induced By TGF-β

[Background] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) on hematopoietic stem progenitor cells (HSPCs) may have some roles in the negative regulation of the HSPC commitment induced by inflammatory cytokines given the fact that progenies of GPI(-) HSPC are often detected in patients with immune-mediated bone marrow (BM) failure. CD109, one of the GPI-APs expressed by keratinocytes and HSPCs in humans, serves as a TGF-β co-receptor and is reported to inhibit TGF-β signaling in keratinocytes; however, the role of CD109 on HSPCs remains unknown. We previously demonstrated that TGF-β induced erythroid differentiation of TF-1 cells, a myeloid leukemia cell line that expresses CD109, in a dose-dependent manner and that knockout of the CD109 gene resulted in erythroid differentiation of TF-1 cells cultured in fetal bovine serum-containing medium, suggesting an inhibitory role of CD109 in the erythroid differentiation of HSPCs induced by low levels of TGF-β (Blood, 2018. 132 (Suppl.1) :3874). However, as most CD109 KO TF-1 cells changed into erythroid cells, they were unsuitable for investigating the role of CD109 in the erythroid differentiation induced by TGF-β. To overcome this issue, we prepared TF-1 cells and cord blood (CB) HSPCs in which the CD109 expression was transiently downregulated, and attempted to further clarify the role of CD109. [Methods] TF-1 cells and CD34+ cells isolated from CB mononuclear cells were treated with siRNA that was complementary to CD109 mRNA. CD109 knockdown cells were cultured for 4 days in serum-free medium supplemented with stem cell factor, thrombopoietin, and erythropoietin with or without TGF-β. In separate experiments, TF-1 cells were treated with phosphatidylinositol-specific phospholipase C (PIPL-C) treatment for 1 hour and were incubated in the presence or absence of TGF-β. CD109 KO TF-1 cells were incubated in serum-free medium (StemPro-34 SFM) for 14 days and their phenotype was determined using flow cytometry (FCM). The erythroid differentiation of the cells was assessed by testing the expression of glycophorin A (GPA) and iron staining. [Results] The down-regulation of CD109 in TF-1 cells by the siRNA treatment increased GPA expression in response to 12 ng/ml of TGF-β from 1.77% to 35.6%. The transient depletion of GPI-APs by PIPL-C also augmented the GPA expression induced by TGF-β from 1.27% to 6.77%. In both BM of healthy individuals and CB, CD109 was more abundantly expressed in Lin-CD34+CD38-CD90+CD45RA- hematopoietic stem cells (HSCs) than in Lin-CD34+CD38-CD90-CD45RA- multipotent progenitors (MPPs) and Lin-CD34+CD38+ HSPCs (Fig. 1). The treatment of CB cells with siRNA reduced the CD109 expression in Lin-CD34+CD38+ cells from 55.9% to 23.1%. TGF-β induced the expression of GPA in Lin-CD34+CD38+CD123-CD45RA- megakaryocyte-erythrocyte progenitor cells (MEPs) of CD109 knockdown cells to a greater degree than the control counterpart (Fig. 2). During 14-day serum-free culture, GPA-positive CD109 KO TF-1 cells died, and similarly to WT TF-1 cells, most surviving CD109 KO TF-1 cells were GPA-negative. TGF-β treatment induced erythroid differentiation in CD109 KO TF-1 cells to a greater degree than in WT TF-1 cells. [Conclusions] CD109 plays a key role in the inhibition of TF-1 erythroid differentiation in response to TGF-β. CD109 may suppress TGF-β signaling, and the lack of CD109 may make PIGA-mutated HSPCs more sensitive to TGF-β, thus leading to the preferential commitment of the mutant erythroid progenitor cells to mature red blood cells in immune-mediated BM failure. Disclosures Yamazaki: Novartis Pharma K.K.: Honoraria; Sanofi K.K.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria. Nakao:Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy.

Aberrant Responsiveness of Erythropoiesis to Iron Deficiency in Polycythemia Vera

Polycythemia vera (PV) presents with iron deficiency at diagnosis, and the mainstay of treatment, i.e. therapeutic phlebotomy, worsens iron deficiency. Iron deficiency is associated with anemia and symptoms of cognitive impairment and fatigue even in the absence of anemia, and patients with low risk PV often suffer from iron deficiency related symptoms How iron deficiency develops in PV patients prior to phlebotomy is not well understood. We previously demonstrated that PV patients exhibit a greater extent of iron deficiency relative to wild type JAK2 patients with other causes of erythrocytosis [Ginzburg Leuk 2018]. We hypothesize that mutated JAK2 leads to aberrant insensitivity of erythropoiesis to iron deficiency. To explore this hypothesis, we first analyzed serum data from iron deficient PV patients (n=14) and blood donors (n=15), normalized for age (51 vs. 42 years, P=0.12) and serum ferritin concentration (22 vs. 22 ng/ml, P=0.95); our data demonstrate that PV patients have significantly lower hepcidin, transferrin saturation, MCV, a higher HCT, and a trend toward higher erythroferrone (ERFE) relative to controls (Fig 1a-1e). Secondly, CD34+ cells were isolated from mononuclear cells and plated with erythropoietin and either 100% or 10% transferrin saturation to mimic iron replete and iron deficient conditions, respectively; differentiation and proliferation were analyzed using flow cytometry. These experiments revealed that although glycophorin A (GPA) and CD36+ cells were decreased in iron deficient relative to iron replete control cells, PV cells continue to proliferate irrespective of iron status (Fig 1f). In addition, only iron deficient control, not PV, cells demonstrated an erythroid lineage specific decrease in proliferation relative to iron replete cells (Fig 1g), demonstrating that iron restriction in PV does not limit erythroid differentiation or proliferation in vitro. Thirdly, we transplanted JAK2 V617F (PV) and wild type (WT) cells into recipient females and placed mice on 35ppm (iron replete (IR)) or 2.5ppm (iron deficient (ID)) diets. IR PV mice exhibited the expected erythrocytosis and decrease in MCV and MCH relative to WT controls (Fig 2a-2d). WT mice on an ID diet exhibited decreased MCV, MCH, and RET-He while PV mice had decreased RBC counts with an increased MCHC (Fig 2a, 2c-2f). These findings demonstrate altered iron regulation in PV erythroblasts with a preference for decreasing RBC count, rather than cellular Hb production, in iron deficiency in vivo. No differences were found in the total number of bone marrow erythroblasts in PV mice relative to WT mice on IR or ID diets. IR and ID PV mice demonstrated splenomegaly relative to WT controls. In addition, IR PV erythroblasts expressed significantly more ERFE relative to WT controls with decreased ERFE expression in ID PV erythroblasts (Fig 2g). Similarly, liver hepcidin expression was lower in IR PV relative to WT controls, but was restored in ID PV mice (Fig 2h), the later likely a response to decreased ERFE expression (Fig 2g); no changes are observed in liver iron concentration in IR relative to ID PV mice. Furthermore, the expected decrease in pSTAT is observed only in ID WT (Fig 2i), not ID PV (Fig 2j) erythroblasts. Lastly, TfR2 protein expression was increased in IR PV relative to WT controls (Fig 2k) and decreased only in ID WT (Fig 2l) but not ID PV (Fig 2m) erythroblasts. Since TfR2 degradation is enhanced during iron deficiency [Khalil JEM 2018] and TfR2 enables iron delivery for mitochondrial heme synthesis [Khalil Blood Adv 2017], persistently increased TfR2 in PV erythroblasts may explain why cellular Hb synthesis (i.e. MCH and RET-He) remains unaffected by iron deficiency in PV mice. Taken together, these findings demonstrate that in vitro iron deficiency fails to limit differentiation and proliferation in PV erythroblasts and enhances STAT5 signaling in ID PV erythroblasts in vivo but results in decreased circulating RBCs in PV mice. In addition, decreased erythroblast ERFE expression in ID PV mice results in increased hepcidin to limit erythroblast iron availability, but persistently increased TfR2 concentration enables mitochondrial iron delivery for Hb synthesis despite cellular iron deficiency. Our studies provide novel mechanistic insights into the dysregulation of iron utilization for erythropoiesis in PV. Disclosures Levine: Prelude Therapeutics: Research Funding; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Research Funding; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria; Novartis: Consultancy; Gilead: Consultancy; Lilly: Honoraria; Loxo: Membership on an entity's Board of Directors or advisory committees; Qiagen: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Isoplexis: Membership on an entity's Board of Directors or advisory committees. Hoffman:Merus: Research Funding. Ginzburg:La Jolla Pharma: Membership on an entity's Board of Directors or advisory committees.

Olfactomedin 4 Inhibits Erythroid Differentiation of Leukemic Cell Lines Induced By TGF-β: A Model of Preferential Commitment of Del(13q) Hematopoietic Stem Cells in Immune-Mediated Bone Marrow Failure

[Background] Olfactomedin 4 (OLFM4), a member of the olfactomedin-related protein family, is constitutively expressed in bone marrow (BM) cells and many gastrointestinal organs and is involved in a variety of cellular functions, including proliferation, differentiation, apoptosis, and cell adhesion in tumor cells. Previous studies have suggested that OLFM4 supports the survival of leukemic stem cells derived from iPS cells of patients with chronic myeloid leukemia. Our recent study revealed a somatic mutation of OLFM4 in HLA allele-lacking granulocytes of a patients with acquired aplastic anemia (AA) in long-term remission (Blood advances, 2(9):1000-1012, 2018). The OLFM4 gene is located at chromosome 13q14.3, which is a commonly deleted region in AA patients with del(13q) who show a good response to immunosuppressive therapy and a high prevalence of increased glycosylphosphatidylinositol-anchored protein(GPI-AP)-deficient cells (Haematologica, 97(12):1845-9, 2012). There may be common mechanisms underlying the preferential commitment between GPI-AP- and del(13q) hematopoietic stem and progenitor cells (HSPCs), like insensitivity to inhibitory cytokines such as TGF-β, in immune-mediated BM failure. To confirm this hypothesis, we studied the effect of OLFM4 knockout on the erythroid differentiation of erythroid leukemia cell lines induced by TGF-β. [Methods] We established OLFM4 knockout (KO) K562 and TF-1 cells using a CRISPR-Cas9 system. OLFM4-knockdown (KD) cells were also prepared using siRNA to validate the results of OLFM4-KO cells. The OLFM4 mRNA and protein levels were determined using quantitative polymerase chain reaction, flow cytometry (FCM), Western blotting, immunocytochemistry, and immunofluorescence methods. The erythroid differentiation was assessed by measuring the expression of glycophorin A (GPA) with FCM, GATA-1 protein expression using Western blotting, and iron staining of the cells. [Results] The OLFM4 KO cells showed slower proliferation than wild-type (WT) cells. Both OLFM4-KO cells and OLFM4-KD cells showed a higher GPA expression than WT cells (median fluorescence intensity [MFI] of K562: 2924 and 2143 vs. 1469 and TF-1: 950 and 870 vs. 694, respectively). OLFM4-KO cells showed erythroid morphology, an elevated expression of GATA-1, and positivity for iron granules, suggesting that OLFM4 KO promoted the erythroid differentiation of K562 and TF-1 cells in RPMI1640 containing 10% fetal bovine serum (Figure 1). When WT cells were cultured in a serum-free culture medium (Steampro34) with or without TGF-β (6 ng/ml) for 8 days, the GPA expression was induced in both TF-1 (MFI: 4358 vs. 883), and K562 cell lines (33440 vs. 25655). The OLFM4 protein levels in these cell lines were significantly decreased by the TGF-β treatment in a dose-dependent manner, suggesting that TGF-β directly downregulated the OLFM4 expression in WT cells; the relative expression of OLFM4 was 1, 0.64, and 0.12 while that of TF-1 was 1, 0.12, and 0.05 at 0, 2, and 6 ng/ml of TGF-β, respectively (Figure 2). [Conclusion] OLFM4 prevents K562 and TF-1 cells from differentiating into erythroid cells in response to TGF-β. The erythroid differentiation of these leukemic cells may be mediated by the downregulation of OLFM4 induced by TGF-β. Haploinsufficiency of OLFM4 due to either a loss of function mutation or del(13q) may be related to the mechanisms underlying the preferential commitment of the mutant HSPCs to erythroid cells in patients with immune-mediated BM failure where TGF-β is abundantly present. Disclosures Yoroidaka: Ono Pharmaceutical: Honoraria. Nakao:Takeda Pharmaceutical Company Limited: Honoraria; Novartis Pharma K.K: Honoraria; Kyowa Kirin: Honoraria; Bristol-Myers Squibb: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ono Pharmaceutical: Honoraria; Celgene: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; SynBio Pharmaceuticals: Consultancy.

Development and Characterization of Monoclonal Antibodies Against Glycophorin A Applicable for Blood Sample Processing.

The separation of plasma from blood cells is critical for the accuracy of blood tests because cellular fractions can create discrepancies in analysis. The most common method to separate blood cells from the liquid part of the blood is centrifugation, which is not always applicable in resource-constrained areas and countries. In this study, we describe the generation of monoclonal antibodies (mAbs) against glycophorin A (GPA) of human erythrocytes. BALB/c mice were immunized with human erythrocytes followed by purified GPA. The splenocytes of the immunized mice were fused with Sp2/0 myeloma cells by hybridoma technique. Hybridoma clones were screened by hemagglutination assay and enzyme-linked immunosorbent assay (ELISA). Six hybridoma clones were obtained and subcloned. The characterization of the purified mAbs demonstrates that they are able to bind and retain erythrocytes in hemagglutination assay. Furthermore, one of the mAbs 1A9 recognizes purified GPA in ELISA, whereas the other mAb 1G7 is able to immunoprecipitate GPA from human erythrocyte lysates, and a band of 38 kDa is detected. In conclusion, the anti-GPA mAbs are useful tools in developing a quick and easy way to separate blood plasma from whole blood for clinical tests, and in developing bi-specific antibodies for other clinical applications.

PB2299 AN INVESTIGATION INTO THE PRESENCE OF COMPLEMENT PROTEINS ON ERYTHROCYTE VESICLES IN SICKLE CELL DISEASE

Background:A single amino acid substitution in the globin gene segment is the underlying genetic defect in sickle cell disease (SCD) creating a haemoglobin molecule which polymerizes on deoxygenation. Recent insights into the pathophysiology of the disease have pointed to the roles inflammation and hypercoagulability play in disease manifestation and severity caused, in part, by increased phosphatidylserine exposure and extracellular vesiculation by sickle red cells.Current works have identified some of these vesicles as those released from reticulocytes in the process of maturation to the red blood cell. These vesicles are termed ‘inside‐out’ autophagic vesicles as they are the result of the fusion of endocytosed parts of the cell membrane with autophagic endosomes and are positive for intracellular glycophorin A (GPA) epitope using BRIC163 antibody. Further analysis has shown that some of these vesicles are also positive for extracellular GPA epitope using BRIC256 antibody raising the possibility that the vesicle membrane may have become permeabilized. One possible permeabilizing mechanism is the membrane attack complex of the complement system.Aims:The aim of this project was to investigate the presence of complement proteins on vesicles isolated from plasma or attached to erythrocytes using flow cytometry and confocal microscopyMethods:Vesicles were isolated from SCD plasma (n = 2) and healthy control (n = 1) using 2 different techniques: membrane‐based isolation technique and fluorescent‐activated cell sorter (FACS) and studied with flow cytometry using BRIC163, BRIC256, C3, C4, C3c and C3d antibodies. Cultured reticulocyte (n = 2), sickle red cells (n = 3) and healthy control red cells (n = 2) were analyzed with confocal microscopy using Annexin‐V, BRIC163, C3, C4, C3c and C3d antibodies.Results:Results show the presence of complement proteins on vesicles in both SCD plasma and healthy control in levels as high as 100% for BRIC163 vesicles in healthy control. Confocal analyses reveal this deposition is also present on vesicles prior to release and is associated with PS exposure.Summary/Conclusion:Complement proteins are present on vesicles in plasma and erythrocytes and may suggest activation and formation of the MAC complex. Further work is required to determine the involvement of these proteins in vesicle formation and release.

Bone marrow characterization in sickle cell disease: inflammation and stress erythropoiesis lead to suboptimal CD34 recovery.

Stress erythropoiesis and chronic inflammation in subjects with sickle cell disease (SCD) may have an impact on the bone marrow (BM) haematopoietic stem and progenitor cell (HSPC) quality and yield necessary for effective autologous, exvivo HSPC gene therapy. BM from 19 subjects with SCD and five volunteers without SCD (non-SCD) was collected in different anticoagulants and processed immediately (day 0) or the following day (day 1). Inflammatory, contamination and aggregation markers within the mononuclear layer, and CD34, CD45 and Glycophorin-A (GPA) expression on HSPCs after CD34+ selection were analysed by conventional and imaging flow cytometry. Compared to non-SCD BM, multiple markers of inflammation, contamination (red cells, P<0·01; platelets, P<0·01) and aggregates (platelet/granulocytes, P<0·01; mononuclear/red cells, P<0·01) were higher in SCD BM. Total CD34+ cell count was lower in SCD BM (P<0·05), however CD34+ count was higher in SCD BM when collected in acid citrate dextrose-A (ACDA) versus heparin (P<0·05). Greater than 50% of CD34+ HSPCs from SCD BM are CD34dim due to higher erythroid lineage expression (P<0·01) as single cell CD34+ CD45+ GPA+ (P<0·01) and CD34+ CD45- GPA+ (P<0·01) HSPCs. SCD BM is characterized by increased inflammation, aggregation and contamination contributing to significant differences in HSPC quality and yield compared to non-SCD BM.

Molecular Detection of Glycophorins A and B Variant Phenotypes and their Clinical Relevance.

Crossover or conversion between the homologous regions of glycophorin A (GYPA) and glycophorin B (GYPB) gives rise to several different hybrid glycophorin genes encoding a number of different glycophorin variant phenotypes which bear low prevalence antigens in the MNS blood group system. GP.Mur is the main glycophorin variant phenotype which causes hemolytic transfusion reaction (HTR) and hemolytic disease of the fetus and newborn (HDFN) in East and Southeast Asians. The detection of glycophorin variant phenotypes using serological methods is limited to phenotyping reagents that are not commercially available. Moreover, the red blood cells used for antibody identification are usually of the GP.Mur phenotype. The current Polymerase Chain Reaction (PCR)-based methods and loop-mediated isothermal amplification (LAMP) are available alternatives to phenotyping that allow for the specific detection of glycophorin variant phenotypes. This review highlights the molecular detection method for glycophorins A and B variant phenotypes and their clinical relevance.

In silico model for glycophorin a (GPA) structure

In silico model for glycophorin a (GPA) structure

Clinical approach after identification of a rare anti-Ena in a prenatal sample

The antigens associated with the MNS blood group system (ISBT 002) are located on glycophorin A (GPA) and glycophorin B (GPB). The most frequently encountered antibodies to antigens in this system by a transfusion medicine service are those directed against M, N, S, and s. Individuals lacking GPA typically have red blood cells that lack M, N, and Ena, whereas those lacking both GPA and GPB lack M, N, and Ena as well as S, s, and U. Such individuals may develop a rare antibody, anti-Ena, directed against determinants on GPA. This antibody is capable of causing hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. This case report describes a pregnant woman found to have anti-Ena. Molecular testing supported an Mk phenotype that was found in several members of her immediate family.

An update on the MNS blood group system

This update of the MNS blood group system (Reid ME. MNS blood group system: a review. Immunohematology 2009;25:95-101) reports three new antigens of the MNS system numbered MNS47, MNS48, and MNS49; new glycophorin (GP) variants associated with silent and weak expression of MNS antigens; and the results of new studies on associations of MNS antigens with band 3, Rh proteins, and malaria. The addition of these three antigens brings the total number of antigens in the MNS system (International Society of Blood Transfusion system 2) to 49.

Shed EBA-175 mediates red blood cell clustering that enhances malaria parasite growth and enables immune evasion.

Erythrocyte Binding Antigen of 175 kDa (EBA-175) has a well-defined role in binding to glycophorin A (GpA) during Plasmodium falciparum invasion of erythrocytes. However, EBA-175 is shed post invasion and a role for this shed protein has not been defined. We show that EBA-175 shed from parasites promotes clustering of RBCs, and EBA-175-dependent clusters occur in parasite culture. Region II of EBA-175 is sufficient for clustering RBCs in a GpA-dependent manner. These clusters are capable of forming under physiological flow conditions and across a range of concentrations. EBA-175-dependent RBC clustering provides daughter merozoites ready access to uninfected RBCs enhancing parasite growth. Clustering provides a general method to protect the invasion machinery from immune recognition and disruption as exemplified by protection from neutralizing antibodies that target AMA-1 and RH5. These findings provide a mechanistic framework for the role of shed proteins in RBC clustering, immune evasion, and malaria.