Hemolytic disease of the newborn due to anti-Jra from a Chinese mother with one novel and one classic heterozygous mutation.

OBJECTIVE: Investigation of a Jr(a-) family samples, identification of the mutant and assessment of the differences of Jr antigen density of the Jr(a-) family members, random adult and newborn individuals' RBCs. BACKGROUND: The anti-Jra antibody is generated when a Jr(a-) individual pregnant or transfused with Jr(a+) blood unit, which can lead to mild-to-moderate hemolytic disease of the foetus and newborn (HDFN) or hemolytic transfusion reaction (HTR). Several mutations had been identified. The anti-Jra caused HDFN is not rare in East Asia, but due to the lack of antibody and molecular background, it is likely to lead missed detection. METHODS AND MATERIALS: One G4P1 woman had been detected as IAT positive during prenatal examination. Suspected as anti-Jra after the laboratory serological testing, the maternal sample was further assessed by molecular analysis. The antigen density was detected by flow cytometry after reacting with anti-Jra serum in family members and the normal individuals. RESULTS: One novel frameshift mutation c.717delC and one previously identified mutation c.706C > T in ABCG2 was identified on proband. The infant haemoglobin(Hb) and bilirubin increased significantly after exchange transfusion and the severe HDFN was relieved. Flow cytometry results showed that the Jra antigens on adult RBCs were significantly less than those on the infant. CONCLUSION: The c.717delC mutation can lead to the shortening of protein ABCG2 in the site of p.Leu307Stop, result in the loss of Jra antigen. The difference in antigen density between adult and infant RBCs may be a possible reason that leads to severe HDFN but not transfusion reaction. Breastfeeding may lead to slower recovery from HDFN.

NLRP3 Inflammasome Simultaneously Involved in Autophagy and Phagocytosis of THP-1 Cells to Clear Aged Erythrocytes.

Autophagy and phagocytosis are two important processes that capture and digest materials found in cellular interiors and exteriors, respectively. Aged red blood cells (RBCs) are cleared by phagocytes in vivo. We focused on determining whether autophagy occurs after phagocytes swallow sunset erythrocytes, and whether the degree of autophagy is related to scavenging ability of phagocytes to erythrocytes. In addition, the ability of NLR family pyrin domain containing protein 3 (NLRP3) inflammasome to regulate erythrocyte clearance by phagocytes and its association with autophagy-related protein 16-like protein 1 (ATG16L1) are confirmed. We constructed a stable and low-NLRP3 expression THP-1 cell line using CRISPR/Cas9 technology. The analysis of erythrocyte clearance and autophagy of THP-1 cells with low NLRP3 expression showed that autophagy changes together when THP-1 engulfs aged RBCs. The occurrence of autophagy was dominated by microtubule-associated protein 1A/1B-light chain 3- (LC3-) associated phagocytosis accompanied by canonical autophagy. A negative correlation exists between the clearance of RBCs by THP-1 cells and the degree of autophagy. Downregulating the expression of NLRP3 in THP-1 cells can simultaneously inhibit the scavenging ability of THP-1 to erythrocytes and the degree of autophagy. In addition, the autophagy inhibitor bafilomycin A1 (BafA1) can enhance the phagocytosis ability of THP-1 to erythrocytes and promote the NLRP3 activation in THP-1 cells, while the autophagy inducer rapamycin inhibits the phagocytosis ability of THP-1 to RBCs and downregulates the NLRP3 activation. Results showed that autophagy and phagocytosis may be dynamic balance processes that can provide sufficient nutrition and energy to cells. Choosing NLRP3 as a target may regulate the phagocytic ability and the degree of autophagy in the meantime. These findings may be a potential strategy for regulating the clearance rate of phagocytes to aged RBCs and the secretion of proinflammatory cytokines to ensure transfusion safety.

OK/basigin expression on red blood cells varies between blood donors and correlates with binding of recombinant Plasmodium falciparum reticulocyte-binding protein homolog 5.

BACKGROUND: Recently, basigin (BSG), which carries OK antigens on red blood cells (RBCs), was reported to be the receptor of the Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRh5). BSG-PfRh5 is the only essential receptor-ligand pair in P. falciparum invasion that is known to date. However, the kind of OK/BSG polymorphism involved in the selection pressure caused by P. falciparum malaria has not been determined. STUDY DESIGN AND METHODS: Blood samples were collected to detect the expression of OK/BSG. The coding region of PfRh5 was cloned and expressed. Enzyme-linked immunosorbent assay-based erythrocyte binding assay was used to measure the recombinant PfRh5 (rPfRh5) binding of RBCs with different OK/BSG expressions. Sequencing of the BSG gene and quantification of the BSG mRNA were performed for selected samples. The candidate microRNAs (miRNAs), which might target the BSG gene, were obtained by miRNA sequencing. Dual-Luciferase reporter assay and overexpression of identified miRNAs were performed in K562 cells. RESULTS: The rPfRh5 was successfully expressed and verified. The OK/BSG expression levels varied among blood donors and were strongly associated with rPfRh5 binding. No single-nucleotide polymorphism was related to the OK/BSG expression. A potential BSG regulator, miR-501-3p, was identified by miRNA sequencing and Dual-Luciferase assay, but was not proven to regulate the expression of BSG in K562 cells. CONCLUSION: Although the mechanism of OK/BSG expression and regulation on RBCs has not been fully clarified, our findings suggest that the OK/BSG expression levels on RBCs might be related to P. falciparum invasion. Moreover, posttranscriptional regulation might play a role in controlling the OK/BSG expression.