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Background: Severe malarial anemia is a leading cause of death in African children under 3 years of age who are infected by Plasmodium(P)falciparum. Pathogenesis of this anemia is not understood. It has been hypothesized that it may be due to the effects of the parasite on host's red blood cells (RBCs), the immune system's battle to fight off the parasite, or dyserythropoiesis. Whether induced by a single or multiple factors, uninfected as well as infected RBCs are rapidly cleared from circulation. The purpose of this study was to determine if P falciparum induces changes in RBC membranes that contributed to the immune destruction of RBCs. Study Design and Methods: RBCs were collected from healthy subjects and tested using standard hemagglutination assays for 45 antigens representing 21 blood group systems and antigen collections before and after exposure to P falciparum, strain FVO. Lectins were used to determine whether crypt or neoantigens were expressed on the RBC membrane. Polybrene was used to detect changes in sialic acid. RBCs were cultured in vitro with and without the parasite and blinded serologic studies were completed. CD35(Complement Receptor 1), CD55(Decay Accelerating Factor), CD59(Membrane Inhibitor of Reactive Lysis) and CD47(Integrin-associated protein) expression was assessed by flow cytometry and compared for infected and uninfected RBCs. Percent parasitemia was determined using Giemsa stained thin blood films. Results: Two (Ch, Lu[sup b]) of 45 antigens tested had differing strengths of agglutination between infected and uninfected RBCs but were resolved with a second source of antisera. 43 antigens showed no significant differences in strength of agglutination between the infected and uninfected RBCs. Lectin and polybrene testing showed no differences. CD35, CD55, CD59 and CD47 levels showed no statistically significant differences. Conclusion: P falciparum does not appear to alter the expression of classified immunogenic antigens on the RBC membrane in this in vitro system. Previously observed changes in CD35, CD55, and CD59 levels on RBCs of infected hosts that were not observed in this study indicate that these protein levels may be controlled by the host immune system. Perhaps evaluating CD47 levels in infected hosts will provide insight into the cause of anemia. Pathogenesis of the hemolytic episode that occurs in these children remains unclear but would appear to be immune mediated. [ABSTRACT FROM AUTHOR]

Background: Alloimmunization to the human neutrophil alloantigen HNA-2a (NB1) plays an important part in alloimmune neonatal neutropenia, immune neutropenia after bone marrow transplantation, drug-induced immune neutropenia and in transfusion-related acute lung injury (TRALI). The NB1 glycoprotein, which has been recently clustered as CD177, is expressed on the neutrophil plasma membrane of 88% to 97% of healthy individuals. In this study, we elucidated the primary structure of the NB1 glycoprotein and the molecular basis of the HNA-2a (NB1)-negative phenotype. Methods: By the use of the well-characterized HNA-2a-specific monoclonal antibody 7D8, we isolated the antigen by immunoaffinity chromatography. The molecular mass was determined by MALDI-TOF mass spectrometry. After elucidation of the N-terminal amino acid sequence by Edman degradation primers we designed primers for rapid amplification of cDNA ends (RACE) PCR. The obtained PCR products were sequenced and COS-7 cells were transfected with the obtained cDNA. In addition, we investigated the neutrophils of two individuals who did not express the HNA-2a (NB1) antigen on their neutrophils. Results: The Mr of the NB1 glycoprotein was found to be 50.6 kDa with a 7.5 kDa carbohydrate moiety. In the obtained 1,614 bp cDNA, a 1,311 bp sequence was identified to represent the entire coding region. Transfected COS-7 cells expressed the HNA-2a (NB1) antigen on their membrane. The DNA sequence encoding a protein of 416 amino acids and a 21 amino acid signal peptide was found to be spread over nine exons in the genome. The deduced amino acid sequence predicted a protein with 3 N-linked carbohydrate binding sites, 2 cysteine-rich domains, and a glycosylphosphatidylinositol (GPI)-attachment (v) site. Homology searches indicated that the NB1 glycoprotein belongs to the urokinase-type plasminogen activator receptor (uPAR)/CD59/Ly-6 or uPAR superfamily. In the genome of two HNA-2a negative individuals, we identified the HNA-2a coding sequence. However, NBl-related mRNAs of the two individuals always showed accessory sequences which were considered to be introns because of the typical acceptor and donator splicing sites. Conclusions: We characterized the neutrophil-specific HNA-2a (NB1) alloantigen as a GPI-anchored 50.6 kDa glycoprotein which belongs to the uPAR/CD59/Ly-6 (uPAR) superfamily. The HNA-2a (NB1)-negative phenotype is most likely the result of an expression defect and not of a gene deletion. [ABSTRACT FROM AUTHOR]