Akkermansia muciniphila exoglycosidases target extended blood group antigens to generate ABO-universal blood.

Matching donor and recipient blood groups based on red blood cell (RBC) surface ABO glycans and antibodies in plasma is crucial to avoid potentially fatal reactions during transfusions. Enzymatic conversion of RBC glycans to the universal group O is an attractive solution to simplify blood logistics and prevent ABO-mismatched transfusions. The gut symbiont Akkermansia muciniphila can degrade mucin O-glycans including ABO epitopes. Here we biochemically evaluated 23 Akkermansia glycosyl hydrolases and identified exoglycosidase combinations which efficiently transformed both A and B antigens and four of their carbohydrate extensions. Enzymatic removal of canonical and extended ABO antigens on RBCs significantly improved compatibility with group O plasmas, compared to conversion of A or B antigens alone. Finally, structural analyses of two B-converting enzymes identified a previously unknown putative carbohydrate-binding module. This study demonstrates the potential utility of mucin-degrading gut bacteria as valuable sources of enzymes for production of universal blood for transfusions.

Truncated glycosyltransferase coding regions in novel ABO alleles give rise to weak A or B blood group expression and discrepant typing results.

BACKGROUND: Correct ABO blood-group matching between donor and patient is crucial for safe transfusions. We investigated the underlying reason causing inconclusive ABO serology in samples referred to our laboratory. STUDY DESIGN AND METHODS: Flow cytometric analysis, ABO genotyping, and sequencing were used to characterize ABO-discrepant blood samples (n = 13). ABO gene variants were inserted in a GFP-containing bicistronic vector to assess A/B expression following overexpression in HeLa cells. RESULTS: Seven novel alleles with nonsense mutations predicted to truncate the encoded ABO glycosyltransferases were identified. While these variants could represent O alleles, serology showed signs of ABO glycosyltransferase activity. ABO*A1.01-related alleles displayed remarkably characteristic percentages of A-positive cells for samples with the same variant: c.42C>A (p.Cys14*; 10%), c.102C>A (p.Tyr34*; 31%-32%, n = 2), c.106dup (p.Val36Glyfs*21; 16%-17%, n = 3) or c.181_182ins (p.Leu61Argfs*21; 12%-13%, n = 2). Transfection studies confirmed significantly decreased A expression compared to wild type. The remaining variants were found on ABO*B.01 background: c.1_5dup (pGly3Trpfs*20), c.15dup (p.Arg6Alafs*51) or c.496del (p.Thr166Profs*26). Although the absence of plasma anti-B was noted overall, B antigen expression was barely detected on erythrocytes. Overexpression confirmed decreased B in two variants compared to wildtype while c.1_5dup only showed a non-significant downward trend. CONCLUSION: Samples displaying aberrant ABO serology revealed seven principally interesting alleles. Despite the presence of truncating mutations, normally resulting in null alleles, low levels of ABO antigens were detectable where alterations affected ABO exons 1-4 but not exon 7. This is compatible with the previously proposed concept that alternative start codons in early exons can be used to initiate the translation of functional ABO glycosyltransferase.

ABO Genotyping finds more A2 to B kidney transplant opportunities than lectin-based subtyping.

ABO compatibility is important for kidney transplantation, with longer waitlist times for blood group B kidney transplant candidates. However, kidneys from non-A1 (eg, A2) subtype donors, which express less A antigen, can be safely transplanted into group B recipients. ABO subtyping is routinely performed using anti-A1 lectin, but DNA-based genotyping is also possible. Here, we compare lectin and genotyping testing. Lectin and genotype subtyping was performed on 554 group A deceased donor samples at 2 transplant laboratories. The findings were supported by 2 additional data sets of 210 group A living kidney donors and 124 samples with unclear lectin testing sent to a reference laboratory. In deceased donors, genotyping found 65% more A2 donors than lectin testing, most with weak lectin reactivity, a finding supported in living donors and samples sent for reference testing. DNA sequencing and flow cytometry showed that the discordances were because of several factors, including transfusion, small variability in A antigen levels, and rare ABO∗A2.06 and ABO∗A2.16 sequences. Although lectin testing is the current standard for transplantation subtyping, genotyping is accurate and could increase A2 kidney transplant opportunities for group B candidates, a difference that should reduce group B wait times and improve transplant equity.

Network pharmacology of triptolide in cancer cells: implications for transcription factor binding.

Background Triptolide is an active natural product, which inhibits cell proliferation, induces cell apoptosis, suppresses tumor metastasis and improves the effect of other therapeutic treatments in several cancer cell lines by affecting multiple molecules and signaling pathways, such as caspases, heat-shock proteins, DNA damage and NF-ĸB. Purpose We investigated the effect of triptolide towards NF-ĸB and GATA1. Methods We used cell viability assay, compare and cluster analyses of microarray-based mRNA transcriptome-wide expression data, gene promoter binding motif analysis, molecular docking, Ingenuity pathway analysis, NF-ĸB reporter cell assay, and electrophoretic mobility shift assay (EMSA) of GATA1. Results Triptolide inhibited the growth of drug-sensitive (CCRF-CEM, U87.MG) and drug-resistant cell lines (CEM/ADR5000, U87.MGΔEGFR). Hierarchical cluster analysis showed six major clusters in dendrogram. The sensitive and resistant cell lines were statistically significant (p = 0.65 × 10-2) distributed. The binding motifs of NF-κB (Rel) and of GATA1 proteins were significantly enriched in regions of 25 kb upstream promoter of all genes. IPA showed the networks, biological functions, and canonical pathways influencing the activity of triptolide towards tumor cells. Interestingly, upstream analysis for the 40 genes identified by compare analysis revealed ZFPM1 (friend of GATA protein 1) as top transcription regulator. However, we did not observe any effect of triptolide to the binding of GATA1 in vitro. We confirmed that triptolide inhibited NF-κB activity, and it strongly bound to the pharmacophores of IκB kinase ß and NF-κB in silico. Conclusion Triptolide showed promising inhibitory effect toward NF-κB, making it a potential candidate for targeting NF-κB.

A novel ABO allele with a 21-bp duplication identified in two unrelated European individuals with weak A expression.

OBJECTIVES: To carry out genetic and serological analyses of a Swiss blood donor and a Danish patient carrying an aberrant ABO phenotype with weak A expression. BACKGROUND: ABO is the most clinically important blood group system but also one of the most complex. The system antigens are determined by carbohydrate structures generated by A and B glycosyltransferases encoded by the ABO gene. Genetic variants of ABO may encode a glycosyltransferase with reduced activity, leading to weak expression of A antigen. METHODS: Samples from two individuals were examined using genetic testing and extended immunohaematological evaluation, including standard serological methods, flow cytometry and analysis of plasma glycosyltransferase activity. RESULTS: Both individuals were serologically determined to be Aweak B. Genetic testing revealed that both were heterozygous for a novel ABO*A1.01-like allele with an in-frame duplication of 21 nucleotides in exon 7 (c.543_563dup), leading to the insertion of seven amino acids (QDVSMRR). Flow cytometric testing of native red blood cells (RBCs) showed very weak A antigen expression. This was in accordance with the enzyme activity test. CONCLUSION: In summary, we describe a novel A allele with a duplication of 21 nucleotides in exon 7 that significantly decreases the enzyme activity and leads to very weak expression of A antigen. (200 words).

May the FORS be with you: a system sequel.

CONCLUSIONS: This article is an update of the review of the FORS system published in Immunohematology in 2017 (Hult AK, Olsson ML. The FORS awakens: review of a blood group system reborn. Immunohematology 2017;33:64-72). This update incorporates the most recently presented knowledge on this still enigmatic system and its genetic, enzymatic, and immunological aspects. Further insight into the genetic variation and allele frequencies of the GBGT1 locus has been reported, and screening studies regarding the prevalence of naturally occurring anti-FORS1 in human plasma have been performed and presented. More basic knowledge on the specificity of the gene product, the Forssman synthase, has been obtained in several detailed studies, and its relation to the homologous ABO gene has been investigated. Taken together, we summarize recently added information about the carbohydrate-based FORS blood group system (International Society of Blood Transfusion number 031).

GBGT1 is allelically diverse but dispensable in humans and naturally occurring anti-FORS1 shows an ABO-restricted pattern.

BACKGROUND: The FORS histo-blood group system was described in 2013 and much remains to be investigated regarding its genetic and immunohematologic characteristics, as well as its clinical importance. While presence of the c.887G>A-mutated GBGT1 gene, which results in FORS1 glycosphingolipid expression on human red blood cells (RBCs), is rare in the populations tested so far, naturally occurring anti-FORS1 in plasma appears common. STUDY DESIGN AND METHODS: The Erythrogene database was utilized to probe genetic variation in GBGT1 among 2504 individuals in the 1000 Genomes Project. We screened 1108 Swedish blood donors for three principally important single-nucleotide polymorphisms (c.363C>A, c.886C>T, and c.887G>A) and selected samples were analyzed further. Screening for naturally occurring anti-FORS1 in plasma from 100 donors was performed using antigen-positive RBCs. RESULTS: We identified 68 GBGT1 alleles, of which three were previously listed blood group alleles. Eight potential null alleles were observed, based on three different nonsense mutations. Four healthy donors were found homozygous for c.363C>A, which truncates the GBGT1-encoded Fs synthase prematurely. This is the first description of human knock-outs for GBGT1. The c.886C>T mutation that alters the same codon (p.Arg296Trp) changed by c.887G>A (p.Arg296Gln) was overexpressed to investigate if it induces the FORS1+ phenotype. However, c.886C>T did not result in synthesis of FORS1. We detected anti-FORS1 in 10% of all donors tested but none in the A1 or A1B groups. CONCLUSION: We have extended the knowledge of GBGT1 variants, allele frequencies, and the characteristics of naturally occurring antibodies in our newest carbohydrate blood group system, FORS. The finding of c.363C>A-homozygous donors indicates that GBGT1 is dispensable.

The FORS awakens: review of a blood group system reborn.

CONCLUSIONS: The presence of the FORS1 antigen on red blood cells was discovered relatively recently, and in 2012, the International Society of Blood Transfusion (ISBT) acknowledged FORS as blood group system number 031. This rare antigen is carried by a glycosphingolipid and formed by elongation of the P antigen. Most people have naturally occurring anti-FORS1 in their plasma. The clinical significance of these antibodies is unknown in the transfusion setting, but they can hemolyze FORS1+ erythrocytes in the presence of complement in vitro. First believed to be part of the ABO system, it was later shown that the gene encoding the glycosyltransferase giving rise to FORS1 expression is GBGT1. This gene had previously been deemed nonfunctional in humans, but a mutation, so far only detected in FORS1+ individuals, restores the enzymatic activity. Tissue distribution of the antigen in FORS1+ individuals has not been studied in detail, although the gene is expressed in several cell types. The antigen itself is known to be a receptor for various pathogens and toxins and has been detected in different forms of cancer, but the implications thereof are not fully understood.

Identification of the Molecular and Genetic Basis of PX2, a Glycosphingolipid Blood Group Antigen Lacking on Globoside-deficient Erythrocytes.

The x2 glycosphingolipid is expressed on erythrocytes from individuals of all common blood group phenotypes and elevated on cells of the rare P/P1/P(k)-negative p blood group phenotype. Globoside or P antigen is synthesized by UDP-N-acetylgalactosamine:globotriaosyl-ceramide 3-ß-N-acetylgalactosaminyltransferase encoded by B3GALNT1. It is the most abundant non-acid glycosphingolipid on erythrocytes and displays the same terminal disaccharide, GalNAcß3Gal, as x2. We encountered a patient with mutations in B3GALNT1 causing the rare P-deficient P1 (k) phenotype and whose pretransfusion plasma was unexpectedly incompatible with p erythrocytes. The same phenomenon was also noted in seven other unrelated P-deficient individuals. Thin-layer chromatography, mass spectrometry, and flow cytometry were used to show that the naturally occurring antibodies made by p individuals recognize x2 and sialylated forms of x2, whereas x2 is lacking on P-deficient erythrocytes. Overexpression of B3GALNT1 resulted in synthesis of both P and x2. Knockdown experiments with siRNA against B3GALNT1 diminished x2 levels. We conclude that x2 fulfills blood group criteria and is synthesized by UDP-N-acetylgalactosamine: globotriaosylceramide 3-ß-N-acetylgalactosaminyltransferase. Based on this linkage, we proposed that x2 joins P in the GLOB blood group system (ISBT 028) and is renamed PX2 (GLOB2). Thus, in the absence of a functional P synthase, neither P nor PX2 are formed. As a consequence, naturally occurring anti-P and anti-PX2 can be made. Until the clinical significance of anti-PX2 is known, we also recommend that rare P1 (k) or P2 (k) erythrocyte units are preferentially selected for transfusion to P(k) patients because p erythrocytes may pose a risk for hemolytic transfusion reactions due to their elevated PX2 levels.

RIFINs are adhesins implicated in severe Plasmodium falciparum malaria.

Rosetting is a virulent Plasmodium falciparum phenomenon associated with severe malaria. Here we demonstrate that P. falciparum-encoded repetitive interspersed families of polypeptides (RIFINs) are expressed on the surface of infected red blood cells (iRBCs), where they bind to RBCs--preferentially of blood group A--to form large rosettes and mediate microvascular binding of iRBCs. We suggest that RIFINs have a fundamental role in the development of severe malaria and thereby contribute to the varying global distribution of ABO blood groups in the human population.

An age-dependent ABO discrepancy between mother and baby reveals a novel A(weak) allele.

BACKGROUND: Discrepancies in ABO grouping arise due to different reasons, posing a threat to patient safety. Underlying causes include mixed-field agglutination after transfusion, chimerism, fetomaternal exchange, or inheritance of unusual alleles resulting in weak A/B antigen expression. Cord blood from the infant of a group A2 B mother typed as group O, H+. Samples were investigated to elucidate this conundrum. STUDY DESIGN AND METHODS: Genomic DNA was analyzed by ABO genotyping and sequencing. Red blood cells (RBCs) were characterized by routine serology and flow cytometry. Glycosyltransferase structure was predicted with 3D-modeling software. RESULTS: The mother genotyped as ABO*A1.01/B.01, and the baby, ABO*A1.01/O.01.01. Sequencing revealed a substitution, 311T>A, in the ABO*A1-like allele, which predicts Ile104Asn. Flow cytometry demonstrated A antigen on the mother's RBCs equivalent to the A2 phenotype while no A was detectable on cord RBCs. However, blood from the 11-month-old child demonstrated markedly increased A expression, likely reflecting initiation of carbohydrate chain branching. CONCLUSION: We unraveled a novel A(weak) allele (ABO*AW.29) in a case of apparent nonmaternity. Residue 104 is far from the catalytic site and may be involved in stabilizing the glycosyltransferase by dimerization. Our data support that the group AB mother's B-transferase stabilizes the altered A-transferase by heterodimerization, exemplifying the allelic enhancement phenomenon.

Do ABO blood group antigens hamper the therapeutic efficacy of mesenchymal stromal cells?

Investigation into predictors for treatment outcome is essential to improve the clinical efficacy of therapeutic multipotent mesenchymal stromal cells (MSCs). We therefore studied the possible harmful impact of immunogenic ABO blood groups antigens - genetically governed antigenic determinants - at all given steps of MSC-therapy, from cell isolation and preparation for clinical use, to final recipient outcome. We found that clinical MSCs do not inherently express or upregulate ABO blood group antigens after inflammatory challenge or in vitro differentiation. Although antigen adsorption from standard culture supplements was minimal, MSCs adsorbed small quantities of ABO antigen from fresh human AB plasma (ABP), dependent on antigen concentration and adsorption time. Compared to cells washed in non-immunogenic human serum albumin (HSA), MSCs washed with ABP elicited stronger blood responses after exposure to blood from healthy O donors in vitro, containing high titers of ABO antibodies. Clinical evaluation of hematopoietic stem cell transplant (HSCT) recipients found only very low titers of anti-A/B agglutination in these strongly immunocompromised patients at the time of MSC treatment. Patient analysis revealed a trend for lower clinical response in blood group O recipients treated with ABP-exposed MSC products, but not with HSA-exposed products. We conclude, that clinical grade MSCs are ABO-neutral, but the ABP used for washing and infusion of MSCs can contaminate the cells with immunogenic ABO substance and should therefore be substituted by non-immunogenic HSA, particularly when cells are given to immunocompentent individuals.

Forssman expression on human erythrocytes: biochemical and genetic evidence of a new histo-blood group system.

In analogy with histo-blood group A antigen, Forssman (Fs) antigen terminates with α3-N-acetylgalactosamine and can be used by pathogens as a host receptor in many mammals. However, primates including humans lack Fs synthase activity and have naturally occurring Fs antibodies in plasma. We investigated individuals with the enigmatic ABO subgroup A(pae) and found them to be homozygous for common O alleles. Their erythrocytes had no A antigens but instead expressed Fs glycolipids. The unexpected Fs antigen was confirmed in structural, serologic, and flow-cytometric studies. The Fs synthase gene, GBGT1, in A(pae) individuals encoded an arginine to glutamine change at residue 296. Gln296 is present in lower mammals, whereas Arg296 was found in 6 other primates, > 250 blood donors and A(pae) family relatives without the A(pae) phenotype. Transfection experiments and molecular modeling showed that Agr296Gln reactivates the human Fs synthase. Uropathogenic E coli containing prsG-adhesin-encoding plasmids agglutinated A(pae) but not group O cells, suggesting biologic implications. Predictive tests for intravascular hemolysis with crossmatch-incompatible sera indicated complement-mediated destruction of Fs-positive erythrocytes. Taken together, we provide the first conclusive description of Fs expression in normal human hematopoietic tissue and the basis of a new histo-blood group system in man, FORS.

ABO blood groups influence macrophage-mediated phagocytosis of Plasmodium falciparum-infected erythrocytes.

Erythrocyte polymorphisms associated with a survival advantage to Plasmodium falciparum infection have undergone positive selection. There is a predominance of blood group O in malaria-endemic regions, and several lines of evidence suggest that ABO blood groups may influence the outcome of P. falciparum infection. Based on the hypothesis that enhanced innate clearance of infected polymorphic erythrocytes is associated with protection from severe malaria, we investigated whether P. falciparum-infected O erythrocytes are more efficiently cleared by macrophages than infected A and B erythrocytes. We show that human macrophages in vitro and mouse monocytes in vivo phagocytose P. falciparum-infected O erythrocytes more avidly than infected A and B erythrocytes and that uptake is associated with increased hemichrome deposition and high molecular weight band 3 aggregates in infected O erythrocytes. Using infected A(1), A(2), and O erythrocytes, we demonstrate an inverse association of phagocytic capacity with the amount of A antigen on the surface of infected erythrocytes. Finally, we report that enzymatic conversion of B erythrocytes to type as O before infection significantly enhances their uptake by macrophages to observed level comparable to that with infected O wild-type erythrocytes. These data provide the first evidence that ABO blood group antigens influence macrophage clearance of P. falciparum-infected erythrocytes and suggest an additional mechanism by which blood group O may confer resistance to severe malaria.

Flow cytometry evaluation of red blood cells mimicking naturally occurring ABO subgroups after modification with variable amounts of function-spacer-lipid A and B constructs.

BACKGROUND: Kodecytes bearing synthetic blood group A and B antigens are increasingly being used in transfusion laboratories as serologic mimics of red blood cell (RBC) A(weak) and B(weak) subtypes. The aim of this study was to compare the flow cytometry profile of kodecytes with native ABO subgroups. STUDY DESIGN AND METHODS: A series of A/B kodecytes, each with decreasing A or B antigen expression, were prepared from group O RBCs that were modified with dilutions of function-spacer-lipid KODE technology (FSL) constructs representing a wide serologic range. Using an established flow cytometry method designed for the detection of low levels of A/B antigens, kodecyte profiles were compared with those of native subgroup cells. RESULTS: Kodecytes with positive tube serology from 4+ to 1+ were created with 15 to 2 µg/mL FSL-A or 78 to 10 µg/mL FSL-B transformation solutions. The kodecytes created with higher concentrations of FSL constructs revealed a uniform and/or even distribution of antigens as seen by a single flow cytometry peak more narrow than the broader peaks produced with lower FSL concentrations similar to those found in native A(x) and most B(weak) subgroups. CONCLUSIONS: Although kodecytes are created artificially, they can be designed to mimic the serologic and flow cytometric profiles of native ABO subgroup RBCs.

Weak A phenotypes associated with novel ABO alleles carrying the A2-related 1061C deletion and various missense substitutions.

BACKGROUND: The 1061delC single-nucleotide polymorphism (SNP) has been reported mostly in the context of the common A(2)[A201] allele and typically produces an A(2) phenotype. This study evaluated new A(weak) alleles, each containing 1061delC. STUDY DESIGN AND METHODS: Twenty samples were referred to our laboratory for analysis due to suspected A(weak) phenotypes originally detected at the referring centers. ABO Exons 1 through 7 and flanking intronic regions were sequenced. A antigen expression on red blood cells was analyzed by flow cytometry. Plasma enzyme activity was studied in one case. Molecular three-dimensional modeling techniques studied the potential effects of amino acid changes on the resulting glycosyltransferases (GTs). RESULTS: Thirteen alleles were discovered, each featuring 1061delC with at least 1 of 12 additional SNPs in the coding region. One of these SNPs disrupts the translation initiation codon. Another constitutes the first reported change in the DVD motif. One SNP found in three alleles causes a substitution of one of the four amino acids that differentiates the wild-type A and B enzymes but plasma enzyme analysis by two methods showed only slightly decreased or normal A(2) activity. Flow cytometric analysis semiquantified the A antigen levels in 16 cases featuring 10 of the alleles and ranged from very weak to nearly A(2) levels. However, the majority of the samples displayed A(x)-like patterns. Molecular modeling of some of the GT variants indicated conformational changes that may explain the diminished A expression observed. CONCLUSION: Missense SNPs were identified in 13 novel A(2)-like alleles, which produced a variety of A subgroup phenotypes.