Red Blood Cell Targets Research Articles

Periconception Red Blood Cell Folate and Offspring Congenital Heart Disease : Nested Case-Control and Mendelian Randomization Studies.

Periconception folic acid supplementation has been suggested to protect against congenital heart disease (CHD), but the association between maternal red blood cell (RBC) folate, the gold-standard biomarker of folate exposure, and subsequent offspring CHD risk is lacking. To quantify the association between periconception maternal RBC folate and offspring CHD risk. Prospective, nested, case-control study and 1-sample Mendelian randomization. (ClinicalTrials.gov: NCT02737644). 29 maternity institutions in 12 districts of Greater Shanghai, China. All 197 mothers of offspring with CHD and 788 individually matched mothers of unaffected offspring from the SPCC (Shanghai Preconception Cohort). Maternal RBC folate was measured before or at early pregnancy. Odds ratios [ORs] were estimated using conditional logistic regression after adjustment for covariates. Mendelian randomization was done using the methylenetetrahydrofolate reductase (MTHFR) C677T as the genetic instrument. Case patients had lower median maternal RBC folate concentrations than control participants (714 nmol/L [interquartile range, 482 to 1008 nmol/L] vs. 788 nmol/L [557 to 1094 nmol/L]). Maternal RBC folate concentrations were inversely associated with offspring CHD (adjusted OR per 100 nmol/L, 0.93 [95% CI, 0.89 to 0.99]). The adjusted OR for mothers with periconception RBC folate of 906 nmol/L or more (vs. <906 nmol/L) was 0.61 (CI, 0.40 to 0.93). Mendelian randomization showed that each 100-nmol increase in maternal RBC folate concentrations was significantly associated with reduced offspring CHD risk (OR, 0.75 [CI, 0.61 to 0.92]). Potential confounding due to unmeasured covariates in the nested case-control study. Higher maternal RBC folate is associated with reduced offspring CHD risk. For primary CHD prevention, higher target RBC folate levels than currently recommended for neural tube defect prevention may be needed and warrant further study. National Key Research and Development Program of China, National Natural Science Foundation of China, China Postdoctoral Science Foundation, and Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences.

Baseline red blood cell and breast milk DHA levels affect responses to standard dose of DHA in lactating women on a controlled feeding diet

BackgroundThe importance of providing the newborn infant with docosahexaenoic acid (DHA) from breast milk is well established. However, women in the United States, on average, have breast milk DHA levels of 0.20%, which is below the worldwide average (and proposed target) of >0.32%. Additionally, the relationship between maternal red blood cell (RBC) and breast milk DHA levels may provide insight into the sufficiency of DHA recommendations during lactation. Whether the standard recommendation of at least 200 mg/day of supplemental DHA during lactation is sufficient for most women to achieve a desirable RBC and breast milk DHA status is unknown. MethodsLactating women (n = 27) at about 5 weeks postpartum were enrolled in a 10–12 week controlled feeding study that included randomization to 480 or 930 mg choline/d (diet plus supplementation). As part of the intervention, all participants were required to consume a 200 mg/d of microalgal DHA. RBC and breast milk DHA levels were measured by capillary gas chromatography in an exploratory analysis. ResultsMedian RBC DHA was 5.0% (95% CI: 4.3, 5.5) at baseline and 5.1% (4.6, 5.4) after 10 weeks of supplementation (P = 0.6). DHA as a percent of breast milk fatty acids increased from 0.19% (0.18, 0.33) to 0.34% (0.27, 0.38) after supplementation (P<0.05). The proportion of women meeting the target RBC DHA level of >5% was unchanged (52% at baseline and week 10). The proportion of women achieving a breast milk DHA level of >0.32% approximately doubled from 30% to 56% (p = 0.06). Baseline RBC and breast milk DHA levels affected their responses to supplementation. Those with baseline RBC and breast milk DHA levels above the median (5% and 0.19%, respectively) experienced no change or a slight decrease in levels, while those below the median had a significant increase. Choline supplementation did not significantly influence final RBC or breast milk DHA levels. ConclusionsOn average, the standard prenatal DHA dose of 200 mg/d did not increase RBC DHA but did increase breastmilk DHA over 10 weeks in a cohort of lactating women in a controlled-feeding study. Baseline DHA levels in RBC and breast milk affected the response to DHA supplementation, with lower levels being associated with a greater increase and higher levels with no change or a slight decrease. Additional larger, dose-response DHA trials accounting for usual intakes and baseline DHA status are needed to determine how to best achieve target breast milk DHA levels and to identify additional modifiers of the variable breast milk DHA response to maternal DHA supplementation.

A modified two-color flow cytometry assay to quantify in-vitro reinvasion and determine invasion phenotypes at low Plasmodium falciparum parasitemia

Invasion of human red blood cells (RBCs) by Plasmodium parasites is a crucial yet poorly characterised phenotype. Two-color flow cytometry (2cFCM) promises to be a very sensitive and high throughput method for phenotyping parasite invasion. However, current protocols require high (~1.0%) parasitemia for assay set-up and need to be adapted for low parasitemia samples, which are becoming increasingly common in low transmission settings. Background fluorescence from nuclei-containing uninfected RBCs and high autologous reinvasion rates (merozoite invasion of donor uninfected RBCs present at 50% assay volume) are some of the limitations to the method's sensitivity to enumerate low parasitemia (<0.5%) with nucleic acid-based stains. Here, we describe modifications for plating unlabeled donor to labeled target RBCs per assay well and for gating parasitemia, that produces accurate quantifications of low reinvasion parasitemia. Plasmodium falciparum 3D7, Dd2 and field isolates at various low and high parasitemia (0.05%–2.0%) were used to set-up SyBr Green 1-based 2cFCM invasion assays. Target RBCs were labeled with CTFR proliferation dye. We show that this dye combination allowed for efficient parasite invasion into target RBCs and that a 1:3 ratio of unlabeled to labeled RBCs per assay greatly skewed autologous reinvasion (p < 0.001). Accuracy of quantifying reinvasion was limited to an assay parasitemia of 0.02% with minimal background interference. Invasion inhibition by enzymatic treatments increased averagely by 10% (p<0.05) across the entire parasitemia range. The effect was greater for samples with <0.5% parasitemia. Overall, a more sensitive method for phenotyping invasion of low P. falciparum parasitemia is described.

Enumeration of the Invasion Efficiency of Plasmodium falciparum In Vitro in Four Different Red Blood Cell Populations Using a Three‐Color Flow Cytometry‐Based Method

A novel in vitro culture system using variable concentrations of biotin/streptavidin to label red blood cells (RBCs) that allows for the simultaneous comparison of growth rates in Plasmodium falciparum malaria parasite in four heterogeneous target RBC populations is described. Donor RBCs containing both P. falciparum-infected RBCs and non-infected RBCs at 0.5% parasitemia were first labeled with 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) succinimidyl ester (DDAO-SE) followed by co-culture with a mixture of equal numbers of four differentially biotin/streptavidin labeled RBC populations. After two to three schizogonic growth cycles, co-cultures were harvested and stained with streptavidin-phycoerythrin (SA-PE) followed by staining of parasite-infected RBCs with nucleic acid fluorochrome SYBR Green I. To demonstrate the application of this method, some target RBC populations that had sialic acid residues removed using neuraminidase treatment were mixed with RBC populations without enzymatic treatment and incubated with donor parasitized RBCs strain W2 (sialic acid-dependent) or 3D7 (sialic acid-independent). Significant less susceptibility to malaria parasite invasion was obtained with enzyme-treated RBC populations when compared with non-treated RBCs in blood samples from the same individual when using malaria parasite strain W2, whereas no difference in percent parasitemias was noted following infection with malaria parasite strain 3D7. This novel malaria culture method is cheap and provides increased sensitivity for direct comparison of parasite growth over time of any of the four RBC populations under identical conditions and eliminates the experimental bias due to contaminated donor RBCs. The application of biotin-labeled RBCs will therefore provide a better understanding of invasion phenotype-specific host-parasite interactions and the extent of complex malaria invasion mechanism. © 2019 International Society for Advancement of Cytometry.

Automated tracking of temporal displacements of a red blood cell obtained by time-lapse digital holographic microscopy.

Red blood cell (RBC) phase images that are numerically reconstructed by digital holographic microscopy (DHM) can describe the cell structure and dynamics information beneficial for a quantitative analysis of RBCs. However, RBCs investigated with time-lapse DHM undergo temporal displacements when their membranes are loosely attached to the substrate during sedimentation on a glass surface or due to the microscope drift. Therefore, we need to develop a tracking algorithm to localize the same RBC among RBC image sequences and dynamically monitor its biophysical cell parameters; this information is helpful for studies on RBC-related diseases and drug tests. Here, we propose a method, which is a combination of the mean-shift algorithm and Kalman filter, to track a single RBC and demonstrate that the optical path length of the single RBC can be continually extracted from the tracked RBC. The Kalman filter is utilized to predict the target RBC position in the next frame. Then, the mean-shift algorithm starts execution from the predicted location, and a robust kernel, which is adaptive to changes in the RBC scale, shape, and direction, is designed to improve the accuracy of the tracking. Finally, the tracked RBC is segmented and parameters such as the RBC location are extracted to update the Kalman filter and the kernel function for mean-shift tracking; the characteristics of the target RBC are dynamically observed. Experimental results show the feasibility of the proposed algorithm.

A transgenic mouse model expressing exclusively human hemoglobin E: Indications of a mild oxidative stress

Hemoglobin (Hb) E (β26 Glu→Lys) is the most common abnormal hemoglobin (Hb) variant in the world. Homozygotes for HbE are mildly thalassemic as a result of the alternate splice mutation and present with a benign clinical picture (microcytic and mildly anemic) with rare clinical symptoms. Given that the human red blood cell (RBC) contains both HbE and excess α-chains along with minor hemoglobins, the consequence of HbE alone on RBC pathophysiology has not been elucidated. This becomes critical for the highly morbid β(E)-thalassemia disease. We have generated transgenic mice exclusively expressing human HbE (HbEKO) that exhibit the known aberrant splicing of β(E) globin mRNA, but are essentially non-thalassemic as demonstrated by RBC α/β (human) globin chain synthesis. These mice exhibit hematological characteristics similar to presentations in human EE individuals: microcytic RBC with low MCV and MCH but normal MCHC; target RBC; mild anemia with low Hb, HCT and mildly elevated reticulocyte levels and decreased osmotic fragility, indicating altered RBC surface area to volume ratio. These alterations are correlated with a mild RBC oxidative stress indicated by enhanced membrane lipid peroxidation, elevated zinc protoporphyrin levels, and by small but significant changes in cardiac function. The C57 (background) mouse and full KO mouse models expressing HbE with the presence of HbS or HbA are used as controls. In select cases, the HbA full KO mouse model is compared but found to be limited due to its RBC thalassemic characteristics. Since the HbEKO mouse RBC lacks an abundance of excess α-chains that would approximate a mouse thalassemia (or a human thalassemia), the results indicate that the observed in vivo RBC mild oxidative stress arises, at least in part, from the molecular consequences of the HbE mutation.

Anti-T haemolysins: the effects of sialic acid removal and 2-aminoethylisothiouronium bromide treatment of erythrocytes on immune lysis

Intravascular haemolytic reactions are reported in red-cell T-activated patients after blood transfusion. The relationship between T antigen antibodies present in normal plasma and these reactions remains unclear. In this study, we assessed the haemolytic activity of T antibodies in vitro in comparison with anti-A/B antibodies. We established a haemolysis assay based on treating target red-blood-cells (RBCs) with 2-aminoethylisothiouronium bromide (AET). Two hundred and seven blood donor sera were analysed for anti-T, anti-A/B haemolysins and anti-T agglutinins. Anti-T haemolysins were found in 4 (1·9%) blood donor sera using a standard haemolysis method and in 174 (84%) samples using AET-treated RBCs. Haemolysis correlated with agglutination titres (P<10(-7) ). With both methods, anti-T haemolysins were much weaker than anti-A and anti-B haemolysins. Gradual desialylation of RBCs showed a correlation between sialic acid level as indicated by agglutination with Sambucus nigra lectin and anti-T mediated haemolysis that was significantly increased (fold 2·4) independently of T antigen expression. These data indicate that, in vitro, anti-T-mediated haemolysis depends primarily on the degree of desialylation of target RBCs. They suggest that the haemolytic activity of T antibodies-containing human sera is usually weak and may only become significant in the very rare setting of a profound desialylation of RBCs.

Reduction of disulfide bonds within anti‐D results in enhanced Fcγ receptor blockade

We have investigated whether chemicals known to disrupt disulfide bonds are capable of altering immunoglobulin anti-D structure resulting in an increased efficacy of the chemically modified anti-D to inhibit Fcgamma receptor (FcgammaR)-mediated phagocytosis. If successful, this would provide a rationale to explore this mechanism of enhancing FcgammaR blockade for future use in immunoglobulin therapies for immune cytopenias. Anti-D that was shown to block 50 percent of the FcgammaR-mediated phagocytosis of opsonized red blood cells (RBCs) using a monocyte monolayer assay (MMA) was combined with two different thiol-containing compounds, dithiothreitol (DTT) or p-toluenesulfonylmethyl mercaptan, with or without treatment with iodoacetamide, and allowed to react. Excess chemical was removed by extensive dialysis. FcgammaR blockade was assessed by MMA with dialyzed, untreated, or chemically treated anti-D using both D+ and D- opsonized target RBCs. Toxicity was determined by fluorescence-activated cell sorting. Aggregates and oligomerization of chemically treated anti-D were examined using gel filtration-high pressure liquid chromatography. Using disulfide-reducing compounds to chemically modify anti-D significantly increases the efficacy of the anti-D to induce an FcgammaR blockade and decrease phagocytosis in vitro of opsonized D+ or D- RBCs. This effect was shown not due to unbound residual chemical, toxicity, or formation of immunoglobulin G oligomers. S-alkylation was required when using low concentrations of reducing compound. Our results demonstrate that irreversible reduction of interchain disulfide bonds within the immunoglobulin anti-D results in a significantly increased efficacy to inhibit FcgammaR-mediated phagocytosis regardless of opsonized target cell. With the use of this strategy, more effective and less expensive immunoglobulin treatment for immune cytopenias such as immune thrombocytopenic purpura or autoimmune hemolytic anemia may be developed.

High Resolution Tertiary and Quaternary Structural Changes in Deoxy and Liganded Hemoglobin E

Hemoglobin (Hb) E (β26 Glu→Lys), known to be more unstable than HbA in vitro, remains an enigma in terms of its contributions to red blood cell (RBC) pathophysiological mechanisms. EE individuals exhibit a mild, chronic anemia while HbE-β thalassemia individuals show a range of clinical manifestations, including high morbidity and death, often resulting from cardiac disease. To determine pathophysiological consequences originating from HbE, we created a transgenic mouse expressing exclusively human HbE with equivalent α- and β-chains [Chen et al., Blood 106(11):892a, 2005]. This mice model exhibited hematological characteristics similar to human EE individuals: microcytic RBC with low MCV, low MCH but normal MCHC; target RBC; mild anemia with low Hb, low HCT, elevated reticulocytes; and decreased osmotic fragility. These RBC parameters correlated with indications of mild RBC oxidative stress. Importantly, the transgenic mouse model allows us to obtain purified HbE for crystal growth and dynamic studies, without the presence of human HbA2 (co-expressed in human HbE RBC and difficult to separate). We now present the low [PDB 1YVQ] and high salt [PDB 1YVT] liganded COHbE structures (1.8Å resolution) at or near physiological pH. The known in vitro instability of oxy HbE and its propensity for oxidation may be explained by the substitution of the β26 side-chain with Lys resulting in a positive charge repulsion with consequential changes to β117, and other key stabilizing residues at the α1β1 interface that impart normal association and oxidative stability to HbA. The specific alterations observed in this microenvironment may be the key to HbE instability, given the findings by Adachi et al. (Biochemistry , 42:10252, 2003) that β116 in normal HbA plays a critical role in stabilizing α1β1 interactions and inhibiting oxidation. Additionally, the difference in water structure observed in each of our COHbE structures from crystals generated under different conditions clearly suggests that resolved water structure is dependent simply upon crystallization conditions, and caution must be taken in interpretation of thermal instability. The 1.6 Å structure of deoxy HbE [PDB 3DUT] (crystallization conditions: pH 6.8, 1.8M ammonium phosphate/ammonium sulfate/citrate) exhibits definitive quaternary change compared to the deoxy HbA structure obtained under similar conditions. These structural findings, explain in part, our recent spectroscopic findings of alterations in oxy and deoxy HbE-nitrite interactions.

Morphology and kinetics of the three distinct phases of red blood cell invasion by Plasmodium falciparum merozoites

The invasion of red blood cells (RBCs) is an essential event in the life cycle of all malaria-causing Plasmodium parasites; however, there are major gaps in our knowledge of this process. Here, we use video microscopy to address the kinetics of RBC invasion in the human malaria parasite Plasmodium falciparum. Under in vitro conditions merozoites generally recognise new target RBCs within 1 min of their release from their host RBC. Parasite entry ensues and is complete on average 27.6 s after primary contact. This period can be divided into two distinct phases. The first is an ∼11 s ‘pre-invasion’ phase that involves an often dramatic RBC deformation and recovery process. The second is the classical ‘invasion’ phase where the merozoite becomes internalised within the RBC in a ∼17 s period. After invasion, a third ‘echinocytosis’ phase commences when about 36 s after every successful invasion a dramatic dehydration-type morphology was adopted by the infected RBC. During this phase, the echinocytotic effect reached a peak over the next 23.4 s, after which the infected RBC recovered over a 5–11 min period. By then the merozoite had assumed an amoeboid-like state and was apparently free in the cytoplasm. A comparison of our data with that of an earlier study of the distantly related primate parasite Plasmodium knowlesi indicated remarkable similarities, suggesting that the kinetics of invasion are conserved across the Plasmodium genus. This study provides a morphological and kinetic framework onto which the invasion-associated physiological and molecular events can be overlaid.

Identification of hemoglobin E by the isopropanol solubility test

Hemoglobin (Hb) E (β26 Glu→Lys) is the most common abnormal hemoglobin (Hb) variant in the world. Homozygotes for HbE are mildly thalassemic as a result of the alternate splice mutation and present with a benign clinical picture (microcytic and mildly anemic) with rare clinical symptoms. Given that the human red blood cell (RBC) contains both HbE and excess α-chains along with minor hemoglobins, the consequence of HbE alone on RBC pathophysiology has not been elucidated. This becomes critical for the highly morbid βE-thalassemia disease. We have generated transgenic mice exclusively expressing human HbE (HbEKO) that exhibit the known aberrant splicing of βE globin mRNA, but are essentially non-thalassemic as demonstrated by RBC α/β (human) globin chain synthesis. These mice exhibit hematological characteristics similar to presentations in human EE individuals: microcytic RBC with low MCV and MCH but normal MCHC; target RBC; mild anemia with low Hb, HCT and mildly elevated reticulocyte levels and decreased osmotic fragility, indicating altered RBC surface area to volume ratio. These alterations are correlated with a mild RBC oxidative stress indicated by enhanced membrane lipid peroxidation, elevated zinc protoporphyrin levels, and by small but significant changes in cardiac function. The C57 (background) mouse and full KO mouse models expressing HbE with the presence of HbS or HbA are used as controls. In select cases, the HbA full KO mouse model is compared but found to be limited due to its RBC thalassemic characteristics. Since the HbEKO mouse RBC lacks an abundance of excess α-chains that would approximate a mouse thalassemia (or a human thalassemia), the results indicate that the observed in vivo RBC mild oxidative stress arises, at least in part, from the molecular consequences of the HbE mutation.

Antibody-dependent, cell-mediated cytotoxicity against human red blood cells: correlation of effector cell type with enzymatic alteration of the target cell surface.

Target cell factors, which contribute to the determination of the effector cell type in an antibody-dependent, cell-mediated cytotoxicity system were studied. Human red blood cells (RBC) were treated with papain and investigated by transmission and scanning electron microscopy (TEM and SEM). Both untreated (native) and treated 0 Rh+ RBC were labeled with 51Cr, sensitized with anti-D immunoglobulin and incubated with unfractionated autologous peripheral blood mononuclear cells. With native RBC, immune lysis was proportional to the number of phagocytes: enrichment of effectors in phagocytes increased lysis, while depletion of phagocytes from effectors decreased lysis. Following papain treatment of target RBC, lysis by unfractionated mononuclear effectors was markedly augmented; since this effect was not diminished by decrease of phagocytes to less than 2%, the augmented lysis was not due to the number of phagocytic effectors. TEM and SEM of enzyme-treated RBC showed spherocytosis with varying degrees of crenation and blurring and irregularities of the cellular membranes. The results suggest that papain-induced alterations in the target RBC rendered them susceptible to lysis by interaction between anti-D antibody and peripheral blood lymphocytes.