Application of RhD Blood Group to Simulate Antibody Identification Test in Immunohematology Education.

BACKGROUND: Immunohematology skill education is an important part of the transfusion medicine professional training. We tried to solve the difficulty of obtaining suitable and sufficient positive samples in the immunohematology education. METHODS: Different identification panels and panel cells were created by RhD-positive red blood cells (RBCs) and RhD-negative RBCs, according to the underlying antibodies. Diluted anti-D reagent was used as simulated plasma for identification. RESULTS: The antibody identification of single antibody with dose-effect and two antibodies present at the same time were successfully simulated. CONCLUSIONS: It is a practical and cheap method for antibody identification training to use RhD blood group, especially when positive samples are short.

Erythrocytes of the common carp are immune sentinels that sense pathogen molecular patterns, engulf particles and secrete pro-inflammatory cytokines against bacterial infection.

Introduction: Red blood cells (RBCs), also known as erythrocytes, are underestimated in their role in the immune system. In mammals, erythrocytes undergo maturation that involves the loss of nuclei, resulting in limited transcription and protein synthesis capabilities. However, the nucleated nature of non-mammalian RBCs is challenging this conventional understanding of RBCs. Notably, in bony fishes, research indicates that RBCs are not only susceptible to pathogen attacks but express immune receptors and effector molecules. However, given the abundance of RBCs and their interaction with every physiological system, we postulate that they act in surveillance as sentinels, rapid responders, and messengers. Methods: We performed a series of in vitro experiments with Cyprinus carpio RBCs exposed to Aeromonas hydrophila, as well as in vivo laboratory infections using different concentrations of bacteria. Results: qPCR revealed that RBCs express genes of several inflammatory cytokines. Using cyprinid-specific antibodies, we confirmed that RBCs secreted tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ). In contrast to these indirect immune mechanisms, we observed that RBCs produce reactive oxygen species and, through transmission electron and confocal microscopy, that RBCs can engulf particles. Finally, RBCs expressed and upregulated several putative toll-like receptors, including tlr4 and tlr9, in response to A. hydrophila infection in vivo. Discussion: Overall, the RBC repertoire of pattern recognition receptors, their secretion of effector molecules, and their swift response make them immune sentinels capable of rapidly detecting and signaling the presence of foreign pathogens. By studying the interaction between a bacterium and erythrocytes, we provide novel insights into how the latter may contribute to overall innate and adaptive immune responses of teleost fishes.

MiR-33a-5p in stored red blood cells regulates genes of innate immune response and promotes inflammation.

BACKGROUND AND OBJECTIVES: Blood transfusion is a common therapeutic procedure in hospitalized patients. Red blood cell (RBC) units undergo various biochemical and morphological changes during storage (storage lesion). miRNAs have been studied intensively regarding cellular metabolic processes, but the effect of miRNAs on blood storage is not well defined. MATERIALS AND METHODS: We performed bioinformatics analysis on the public data set of miRNA expression of RBC based on R language, and performed the Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis on the target genes of differentially expressed miRNA. The expression of miRNA differential genes in blood samples stored at different times was verified by qRT-PCR. Next, we used ELISA and qRT-PCR to verify the expression of IL-1ß, IL-6, IL-12 and TNF-α in blood at day 1 and day 42. In addition, in vitro, we transfected macrophages with overexpressed miRNA, and the effects of overexpressed miRNA on macrophage polarization and the release of inflammatory factors were verified by flow cytometry and qRT-PCR and ELISA. RESULTS: This study combined bioinformatics analysis and experiments to discover the differentially expressed miRNAs in long-term stored blood. The results showed that compared to fresh blood samples, the inflammatory factors were significantly doubled by ELISA, as well as the higher mRNA expression at 42 day. Experimentally verified that miR-33a-5p promoted the M1 type macrophage polarization and increased the release of related inflammatory factors through PPARα/ACC2/AMPK/CPT-1a axis regulation. CONCLUSIONS: This study elucidates a potential mechanism of inflammatory factor accumulation in long-term stored blood, providing a theoretical basis and a potential target to prevent transfusion-related adverse reactions.

Circadian Control of the Response of Macrophages to Plasmodium Spp.-Infected Red Blood Cells.

Malaria is a serious vector-borne disease characterized by periodic episodes of high fever and strong immune responses that are coordinated with the daily synchronized parasite replication cycle inside RBCs. As immune cells harbor an autonomous circadian clock that controls various aspects of the immune response, we sought to determine whether the intensity of the immune response to Plasmodium spp., the parasite causing malaria, depends on time of infection. To do this, we developed a culture model in which mouse bone marrow-derived macrophages are stimulated with RBCs infected with Plasmodium berghei ANKA (iRBCs). Lysed iRBCs, but not intact iRBCs or uninfected RBCs, triggered an inflammatory immune response in bone marrow-derived macrophages. By stimulating at four different circadian time points (16, 22, 28, or 34 h postsynchronization of the cells' clock), 24-h rhythms in reactive oxygen species and cytokines/chemokines were found. Furthermore, the analysis of the macrophage proteome and phosphoproteome revealed global changes in response to iRBCs that varied according to circadian time. This included many proteins and signaling pathways known to be involved in the response to Plasmodium infection. In summary, our findings show that the circadian clock within macrophages determines the magnitude of the inflammatory response upon stimulation with ruptured iRBCs, along with changes of the cell proteome and phosphoproteome.

Monocytes, particularly nonclassical ones, lose their opsonic and nonopsonic phagocytosis capacity during pediatric cerebral malaria.

Introduction: Innate immunity is crucial to reducing parasite burden and contributing to survival in severe malaria. Monocytes are key actors in the innate response and, like macrophages, are plastic cells whose function and phenotype are regulated by the signals from the microenvironment. In the context of cerebral malaria (CM), monocyte response constitutes an important issue to understand. We previously demonstrated that decreased percentages of nonclassical monocytes were associated with death outcomes in CM children. In the current study, we postulated that monocyte phagocytosis function is impacted by the severity of malaria infection. Methods: To study this hypothesis, we compared the opsonic and nonopsonic phagocytosis capacity of circulant monocytes from Beninese children with uncomplicated malaria (UM) and CM. For the CM group, samples were obtained at inclusion (D0) and 3 and 30 days after treatment (D3, D30). The phagocytosis capacity of monocytes and their subsets was characterized by flow cytometry and transcriptional profiling by studying genes known for their functional implication in infected-red blood cell (iRBC) elimination or immune escape. Results: Our results confirm our hypothesis and highlight the higher capacity of nonclassical monocytes to phagocyte iRBC. We also confirm that a low number of nonclassical monocytes is associated with CM outcome when compared to UM, suggesting a mobilization of this subpopulation to the cerebral inflammatory site. Finally, our results suggest the implication of the inhibitory receptors LILRB1, LILRB2, and Tim3 in phagocytosis control. Discussion: Taken together, these data provide a better understanding of the interplay between monocytes and malaria infection in the pathogenicity of CM.

Comparison of the Detection Rate and Specificity of Irregular Red Blood Cell Antibodies Between First-Time Pregnant Women and Women With a History of Multiple Pregnancies Among 18,010 Chinese Women.

Background: There is insufficient evidence to assess the risk of the production of clinically important alloimmune irregular red blood cell (RBC) antibodies in first-time pregnant women. Methods: Using the microcolumn gel antiglobulin method, 18,010 Chinese women with a history of pregnancy and pregnant women were screened for irregular RBC antibodies, and for those with positive test results, antibody specificity was determined. The detection rate and specificity of irregular RBC antibodies in women with a history of multiple pregnancies (two or more) and first-time pregnant women were determined. Results: In addition to 25 patients who passively acquired anti-D antibodies via an intravenous anti-D immunoglobulin injection, irregular RBC antibodies were detected in 121 (0.67%) of the 18,010 women. Irregular RBC antibodies were detected in 93 (0.71%) of the 13,027 women with a history of multiple pregnancies, and antibody specificity was distributed mainly in the Rh, MNSs, Lewis, and Kidd blood group systems; irregular RBC antibodies were detected in 28 (0.56%) of the 4983 first-time pregnant women, and the antibody specificity was distributed mainly in the MNSs, Rh, and Lewis blood group systems. The difference in the percentage of patients with irregular RBC antibodies between the two groups was insignificant (χ 2 = 1.248, P > 0.05). Of the 121 women with irregular RBC antibodies, nine had anti-Mur antibodies, and one had anti-Dia antibodies; these antibodies are clinically important but easily missed because the antigenic profile of the reagent RBCs that are commonly used in antibody screens does not include the antigens that are recognized by these antibodies. Conclusion: Irregular RBC antibody detection is clinically important for both pregnant women with a history of multiple pregnancies and first-time pregnant women. Mur and Dia should be included in the antigenic profile of reagent RBCs that are used for performing antibody screens in the Chinese population.

A patient with anti-f in India identified by extensive immunohematologic workup.

Anti-f is produced by exposure to the compound antigen ce (f) on red blood cells (RBCs), expressed when both c and e are present on the same protein (cis position). Although anti-f was discovered in 1953, there are few cases reported worldwide because the presence of anti-f is often masked by anti-c or anti-e and is not generally found as a single antibody. In the present case, anti-f was identified by using three-cell screening and 11-cell identification panels. The identification of anti-f was further supported by additional testing, including (1) Rh antigen typing; (2) antibody identification panels (enzyme-treated panel [ficin] and an in-house-constructed Rh panel); (3) look-back and phenotyping of donor RBC units, which were responsible for alloimmunization; and (4) molecular testing of the patient's RBCs.

Prevalence of Red Blood Cell Alloimmunization Among Pediatric Patients With Sickle Cell Disease in Saudi Arabia.

OBJECTIVE: Sickle cell disease (SCD) is a common hereditary hemoglobin disorder worldwide. One of the main treatments for patients with SCD is the requirement for blood transfusions. Posttransfusion alloimmunization with red blood cell (RBC) antigens continues to be a major risk factor for SCD. The objective of this study was to determine the rate, nature, and risk factors of red cell alloimmunization among pediatric patients with SCD in our center and compare our results with published reports from Saudia Arabia SA, regional countries, and some international countries. MATERIALS AND METHODS: A retrospective chart review of patients with SCD at King Abdulaziz Medical City-Jeddah, between 2008 and 2019 was performed. Demographic characteristics and transfusion histories were recorded. Blood samples were analyzed for alloimmunization using immunohematologic techniques. RESULTS: In total, 121 patients were analyzed. Alloantibodies were detected in 21 patients (17.4%) and were mostly single in 15 patients (71.4%), anti-K (23.7%), anti-E (19.0%), and anti-S (9.5%). The other 6 patients (28.6%) had multiple alloantibodies, especially the combination of anti-C and anti-K (9.5%) and the combination of anti-C and anti-E (9.5%). Alloantibody levels were significantly higher in patients with frequent hospital admissions (>5 times annually), those who had an exchange blood transfusion, those younger than 3 years old, and those who received a larger number of blood units ( P ≤0.05). CONCLUSION: The rate of RBC alloimmunization is determined and considered relatively low compared with that in other nations. Matching for extended RBC antigens to include ABO, RH (D, C, c, E, e), K, Fy a , Fy b , Jk a , and Jk b antigens in the screening panel for donors and recipients is highly recommended to ensure better transfusion practices and avoid transfusion-related complications.

Immunization with PfGBP130 generates antibodies that inhibit RBC invasion by P. falciparum parasites.

Background: Despite decades of effort, Plasmodium falciparum malaria remains a leading killer of children. The absence of a highly effective vaccine and the emergence of parasites resistant to both diagnosis as well as treatment hamper effective public health interventions. Methods and results: To discover new vaccine candidates, we used our whole proteome differential screening method and identified PfGBP130 as a parasite protein uniquely recognized by antibodies from children who had developed resistance to P. falciparum infection but not from those who remained susceptible. We formulated PfGBP130 as lipid encapsulated mRNA, DNA plasmid, and recombinant protein-based immunogens and evaluated the efficacy of murine polyclonal anti-PfGBP130 antisera to inhibit parasite growth in vitro. Immunization of mice with PfGBP130-A (aa 111-374), the region identified in our differential screen, formulated as a DNA plasmid or lipid encapsulated mRNA, but not as a recombinant protein, induced antibodies that inhibited RBC invasion in vitro. mRNA encoding the full ectodomain of PfGBP130 (aa 89-824) also generated parasite growth-inhibitory antibodies. Conclusion: We are currently advancing PfGBP130-A formulated as a lipid-encapsulated mRNA for efficacy evaluation in non-human primates.

The Variation of RhD Blood Group in Different Periods in a Patient with Systemic Lupus Erythematosus.

BACKGROUND: Autoimmune hemolytic anemia disease often produces a large number of various autoantibodies, and some autoantibodies may be related to Rh blood group. In rare cases, autoantibodies can specifically target Rh antigen, thus interfering with the identification of Rh blood group. METHODS: A case of systemic lupus erythematosus (SLE) with inconsistent RhD blood group identification results in different periods was reported and the reasons were analyzed. RESULTS: Some autoantibodies can completely block D antigen on red blood cells, resulting in no redundant D sites on red blood cells binding to reagent anti D. In addition, the immunity of the body is extremely low, and the expression of red blood cell blood group antigens in part of the body is inhibited, which will cause the weakening of the expression of Rh antigen in red blood cells. Therefore, when testing the RhD blood type of the patient, the reagent anti D does not agglutinate with the patient's red blood cells, and a false negative result of the initial screening appears. Through the RhD negative confirmation test, the patient's blood type is a serologically weak D phenotype. CONCLUSIONS: If the result of serological preliminary screening test is RhD negative or RhD variant, the recipient should be treated as RhD negative, and RhD negative red blood cells should be transfused during blood transfusion. Conditional laboratories can implement RHD genotyping, which is conducive to improving the precise blood transfusion management level of RhD negative blood recipients, saving rare blood resources and improving the treatment efficiency of patients.

When and why is red blood cell genotyping applicable in transfusion medicine: a systematic review of the literature.

This review aims to provide a better understanding of when and why red blood cell (RBC) genotyping is applicable in transfusion medicine. Articles published within the last 8 years in peer-reviewed journals were reviewed in a systematic manner. RBC genotyping has many applications in transfusion medicine including predicting a patient's antigen profile when serologic methods cannot be used, such as in a recently transfused patient, in the presence of autoantibody, or when serologic reagents are not available. RBC genotyping is used in prenatal care to determine zygosity and guide the administration of Rh immune globulin in pregnant women to prevent hemolytic disease of the fetus and newborn. In donor testing, RBC genotyping is used for resolving ABO/D discrepancies for better donor retention or for identifying donors negative for high-prevalence antigens to increase blood availability and compatibility for patients requiring rare blood. RBC genotyping is helpful to immunohematology reference laboratory staff performing complex antibody workups and is recommended for determining the antigen profiles of patients and prospective donors for accurate matching for C, E, and K in multiply transfused patients. Such testing is also used to determine patients or donors with variant alleles in the Rh blood group system. Information from this testing aides in complex antibody identification as well as sourcing rare allele-matched RBC units. While RBC genotyping is useful in transfusion medicine, there are limitations to its implementation in transfusion services, including test availability, turn-around time, and cost.

Infection vs. Reinfection: The Immunomodulation of Erythropoiesis.

Severe malarial anemia (SMA) increases the morbidity and mortality of Plasmodium, the causative agent of malaria. SMA is mainly developed by children and pregnant women in response to the infection. It is characterized by ineffective erythropoiesis caused by impaired erythropoietin (EPO) signaling. To gain new insights into the pathogenesis of SMA, we investigated the relationship between the immune system and erythropoiesis, conducting comparative analyses in a mouse model of malaria. Red blood cell (RBC) production was evaluated in infected and reinfected animals to mimic endemic occurrences. Higher levels of circulating EPO were observed in response to (re)infection. Despite no major differences in bone marrow erythropoiesis, compensatory mechanisms of splenic RBC production were significantly reduced in reinfected mice. Concomitantly, a pronounced immune response activation was observed in erythropoietic organs of reinfected animals in relation to single-infected mice. Aged mice were also used to mimic the occurrence of malaria in the elderly. The increase in symptom severity was correlated with the enhanced activation of the immune system, which significantly impaired erythropoiesis. Immunocompromised mice further support the existence of an immune-shaping regulation of RBC production. Overall, our data reveal the strict correlation between erythropoiesis and immune cells, which ultimately dictates the severity of SMA.

[The Identification Idea of Antibodies Against Ku and Other High-Frequency Antigens].

OBJECTIVE: This study was aimed to provide ideas for identifying the antibodies to high-frequency antigens by analyzing a female case of high-frequency antigen antibody (anti-Ku) using serological and sequencing method. METHODS: The methods for identification of blood group, erythrocyte antigen, screening and identification of antibody were used to detect the blood type and antibody in the proband. The proband's serum and reagent screening cells treated with Sulfhydryl reagent were applied to judge the type and characteristics of this antibodies when reacted with the regaent screening cells or proband's serum respectively. Gene sequencing was used to determine the genotype of the proband's blood group. RESULTS: The proband's red blood cells were determined as O type RhD positive, whose serum showed strong positive reaction to antibody-screening cells and antibody identification cells with the same intensity in saline and IAT medium, however, the self-cells showed negative effect. The Direct Antihuman Globulin of proband's red blood cells also showed weak positive reaction, and the other blood types were CcEe, Jk(a+b-), P1-, Le(a-b -), Lu (a-b +), K-, k-, Kp(a-b-). Serum of the proband treated with 2-ME still react with three groups of screening cells in IAT medium. The reaction intensity of proband's serum was also unchanged with the cells modified with papain and bromelain, but showed negative effect when the cells were treated with sulfhydryl agents including DTT and 2-ME. Gene sequencing revealed that the KEL genotype of the patient was KEL*02N.24 . This patient had a rare K0 phenotype. CONCLUSION: The rare Kell-null blood group (also known as K0) were identified by serological and molecular tests in the proband who produced both IgG and IgM type of antibody to high-frequency antigen (anti-Ku). These two methods are of great significance in the identification of this rare blood group as well as the antibody to high frequency antigen.

Breadth of Fc-mediated effector function correlates with clinical immunity following human malaria challenge.

Malaria is a life-threatening disease of global health importance, particularly in sub-Saharan Africa. The growth inhibition assay (GIA) is routinely used to evaluate, prioritize, and quantify the efficacy of malaria blood-stage vaccine candidates but does not reliably predict either naturally acquired or vaccine-induced protection. Controlled human malaria challenge studies in semi-immune volunteers provide an unparalleled opportunity to robustly identify mechanistic correlates of protection. We leveraged this platform to undertake a head-to-head comparison of seven functional antibody assays that are relevant to immunity against the erythrocytic merozoite stage of Plasmodium falciparum. Fc-mediated effector functions were strongly associated with protection from clinical symptoms of malaria and exponential parasite multiplication, while the gold standard GIA was not. The breadth of Fc-mediated effector function discriminated clinical immunity following the challenge. These findings present a shift in the understanding of the mechanisms that underpin immunity to malaria and have important implications for vaccine development.

What is the role of complement in bystander hemolysis? Old concept, new insights.

INTRODUCTION: Bystander hemolysis occurs when antigen-negative red blood cells (RBCs) are lysed by the complement system. Many clinical entities including passenger lymphocyte syndrome, hyperhemolysis following blood transfusion, and paroxysmal nocturnal hemoglobinuria are complicated by bystander hemolysis. AREAS COVERED: The review provides data about the role of the complement system in the pathogenesis of bystander hemolysis. Moreover, future perspectives on the understanding and management of this syndrome are described. EXPERT OPINION: Complement system can be activated via classical, alternative, and lectin pathways. Classical pathway activation is mediated by antigen-antibody (autoantibodies and alloantibodies against autologous RBCs, infectious agents) complexes. Alternative pathway initiation is triggered by heme, RBC microvesicles, and endothelial injury that is a result of intravascular hemolysis. Thus, C5b is formed, binds with C6-C9 compomers, and MAC (C5b-9) is formulated in bystander RBCs membranes, leading to cell lysis. Intravascular hemolysis, results in activation of the alternative pathway, establishing a vicious cycle between complement activation and bystander hemolysis. C5 inhibitors have been used effectively in patients with hyperhemolysis syndrome and other entities characterized by bystander hemolysis.

Impact of transcription factors KLF1 and GATA1 on red blood cell antigen expression: a review.

KLF transcription factor 1 (KLF1) and GATA binding protein 1 (GATA1) are transcription factors (TFs) that initiate and regulate transcription of the genes involved in erythropoiesis. These TFs possess DNA-binding domains that recognize specific nucleotide sequences in genes, to which they bind and regulate transcription. Variants in the genes that encode either KLF1 or GATA1 can result in a range of hematologic phenotypes-from benign to severe forms of thrombocytopenia and anemia; they can also weaken the expression of blood group antigens. The Lutheran (LU) blood group system is susceptible to TF gene variations, particularly KLF1 variants. Individuals heterozygous for KLF1 gene variants show reduced Lutheran antigens on red blood cells that are not usually detected by routine hemagglutination methods. This reduced antigen expression is referred to as the In(Lu) phenotype. For accurate blood typing, it is important to distinguish between the In(Lu) phenotype, which has very weak antigen expression, and the true Lunull phenotype, which has no antigen expression. The International Society of Blood Transfusion blood group allele database registers KLF1 and GATA1 variants associated with modified Lutheran expression. Here, we review KLF1 and recent novel gene variants defined through investigating blood group phenotype and genotype discrepancies or, for one report, investigating cases with unexplained chronic anemia. In addition, we include a review of the GATA1 TF, including a case report describing the second GATA1 variant associated with a serologic Lu(a-b-) phenotype. Finally, we review both past and recent reports on variations in the DNA sequence motifs on the blood group genes that disrupt the binding of the GATA1 TF and either remove or reduce erythroid antigen expression. This review highlights the diversity and complexity of the transcription process itself and the need to consider these factors as an added component for accurate blood group phenotyping.

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.

The influence of neonatal BCG vaccination on in vitro cytokine responses to Plasmodium falciparum.

BACKGROUND: Bacillus Calmette-Guérin (BCG) vaccination has off-target protective effects against infections unrelated to tuberculosis. Among these, murine and human studies suggest that BCG vaccination may protect against malaria. We investigated whether BCG vaccination influences neonatal in vitro cytokine responses to Plasmodium falciparum. Blood samples were collected from 108 participants in the Melbourne Infant Study BCG for Allergy and Infection Reduction (MIS BAIR) randomised controlled trial (Clinical trials registration NCT01906853, registered July 2013), seven days after randomisation to neonatal BCG (n = 66) or no BCG vaccination (BCG-naïve, n = 42). In vitro cytokine responses were measured following stimulation with P. falciparum-infected erythrocytes (PfIE) or E. coli. RESULTS: No difference in the measured cytokines were observed between BCG-vaccinated and BCG-naïve neonates following stimulation with PfIE or E. coli. However, age at which blood was sampled was independently associated with altered cytokine responses to PfIE. Being male was also independently associated with increased TNF-a responses to both PfIE and E. coli. CONCLUSION: These findings do not support a role for BCG vaccination in influencing in vitro neonatal cytokine responses to P. falciparum. Older neonates are more likely to develop P. falciparum-induced IFN-γ and IFN-γ-inducible chemokine responses implicated in early protection against malaria and malaria pathogenesis.

Extracellular vesicles derived from plasmodium-infected red blood cells alleviate cerebral malaria in plasmodium berghei ANKA-infected C57BL/6J mice.

RTS,S is the first malaria vaccine recommended for implementation among young children at risk. However, vaccine efficacy is modest and short-lived. To mitigate the risk of cerebral malaria (CM) among children under the age of 5, it is imperative to develop new vaccines. EVs are potential vaccine candidates as they obtain the ability of brain-targeted delivery and transfer plasmodium antigens and immunomodulators during infections. This study extracted EVs from BALB/c mice infected with Plasmodium yoelii 17XNL (P.y17XNL). C57BL/6J mice were intravenously immunized with EVs (EV-I.V. + CM group) or subcutaneously vaccinated with the combination of EVs and CpG ODN-1826 (EV + CPG ODN-S.C. + CM group) on days 0 and 20, followed by infection with Plasmodium berghei ANKA (P.bANKA) on day 20 post-second immunization. We monitored Parasitemia and survival rate. The integrity of the Blood-brain barrier (BBB) was examined using Evans blue staining.The levels of cytokines and adhesion molecules were evaluated using Luminex, RT-qPCR, and WB. Brain pathology was evaluated by hematoxylin and eosin and immunohistochemical staining. The serum levels of IgG, IgG1, and IgG2a were analyzed by enzyme-linked immunosorbent assay. Compared with those in the P.bANKA-infected group, parasitemia increased slowly, death was delayed (day 10 post-infection), and the survival rate reached 75 %-83.3 % in the EV-I.V. + ECM and EV + CPG ODN-S.C. + ECM groups. Meanwhile, compared with the EV + CPG ODN-S.C. + ECM group, although parasitemia was almost the same, the survival rate increased in the EV-I.V. + ECM group.Additionally, EVs immunization markedly downregulated inflammatory responses in the spleen and brain and ameliorated brain pathological changes, including BBB disruption and infected red blood cell (iRBC) sequestration. Furthermore, the EVs immunization group exhibited enhanced antibody responses (upregulation of IgG1 and IgG2a production) compared to the normal control group. EV immunization exerted protective effects, improving the integrity of the BBB, downregulating inflammation response of brain tissue, result in reduces the incidence of CM. The protective effects were determined by immunological pathways and brain targets elicited by EVs. Intravenous immunization exhibited better performance than subcutaneous immunization, which perhaps correlated with EVs, which can naturally cross BBB to play a better role in brain protection.

A cold case of hemolytic disease of the fetus and newborn resolved by genomic sequencing and population studies to define a new antigen in the Rh system.

BACKGROUND: We report an obstetric case involving an RhD-positive woman who had developed a red blood cell (RBC) antibody that was not detected until after delivery of a newborn, who presented with a positive direct antiglobulin test result. Immunohematology studies suggested that the maternal antibody was directed against a low-prevalence antigen on the paternal and newborn RBCs. RESULTS: Comprehensive blood group profiling by targeted exome sequencing revealed a novel nonsynonymous single nucleotide variant (SNV) RHCE c.486C>G (GenBank MZ326705) on the RHCE*Ce allele, for both the father and newborn. A subsequent genomic-based study to profile blood groups in an Indigenous Australian population revealed the same SNV in 2 of 247 individuals. Serology testing showed that the maternal antibody reacted specifically with RBCs from these two individuals. DISCUSSION: The maternal antibody was directed against a novel antigen in the Rh blood group system arising from an RHCE c.486C>G variant on the RHCE*Ce allele linked to RHD*01. The variant predicts a p.Asn162Lys change on the RhCE protein and has been registered as the 56th antigen in the Rh system, ISBT RH 004063. CONCLUSION: This antibody was of clinical significance, resulting in a mild to moderate hemolytic disease of the fetus and newborn (HDFN). In the past, the cause of such HDFN cases may have remained unresolved. Genomic sequencing combined with population studies now assists in resolving such cases. Further population studies have potential to inform the need to design population-specific red cell antibody typing panels for antibody screening in the Australian population.