Mouse Red Blood Cells Research Articles

High fat diets modulate nitric oxide biosynthesis and antioxidant defence in red blood cells from C57BL/6 mice

The consumption of a high fat (HF) diet is considered a risk factor for the development of obesity. On the other hand, a monounsaturated HF diet has beneficial cardiometabolic effects. Since nitric oxide (NO) modulates vascular homeostasis, we investigate whether HF diets that vary in fatty acid composition have a different effect on the l-arginine-NO pathway and oxidative stress in C57BL/6 mice red blood cells (RBC). The olive oil diet induced an activation of l-arginine transport compared to other diets. NO synthase (NOS) activity was increased in all unsaturated HF diets (olive, sunflower and canola oils). Moreover, the expression of endothelial NOS (eNOS) and inducible NOS (iNOS) was increased in the olive oil group. In contrast, NOS activity from the lard group was decreased associated with diminished l-arginine transport. Olive oil also induced superoxide dismutase activation. Inhibition of the l-arginine-NO pathway in the lard group could contribute to cardiovascular diseases, while unsaturated HF diets may have a protector effect via enhanced NO bioavailability.

Brief report: a new profile of terminal N-acetyllactosamines glycans on pig red blood cells and different expression of α-galactose on Sika deer red blood cells and nucleated cells

It has been reported that: (1) large variations were found in the number of sialic acid (SA) capped with N-acetyllactosamines (SA-Galbeta1-4GlcNAc-R) and alpha-Gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R) or uncapped N-acetyllactosamines (Galbeta1-4GlcNAc-R) on different mammalian red blood cells, and on nucleated cells originating from a given tissue in various species; (2) goat, sheep, horse and mouse red blood cells lack alpha-Gal epitopes, despite the expression of this epitope on a variety of nucleated cells in these species, including lymphocytes differentiated from the same hematopoietic origin. In this study, flow cytometry and Western blot analyses of pig red blood cells showed that alpha-Gal epitopes on pig red cells developed concomitantly after treatment with neuraminidase, suggesting that the terminal N-acetyllactosaminide glycans were capped with SA-alpha-Gal epitopes. Whereas, the expression of the alpha-Gal epitopes on red blood cells from Sika deer (Cevus nippon hortulorum) were found to be absent even though the epitopes were present on their white blood cells. Thus, these results add new data not only for the terminal carbohydrate structures on cell surface glycans of various mammalian cells, but also for wide variety of epitope expression on the cells from different tissues, which might be useful for understanding their unique states resulting from differentiation and evolution.

In situ targeting of dendritic cells by antigen-loaded red blood cells: A novel approach to cancer immunotherapy

Red blood cells (RBCs) were shown to be efficient antigen carriers to target dendritic cells (DCs) and induce cytotoxic T-cell responses. Mouse RBCs were loaded with ovalbumin (RBC-OVA) and injected with Poly (I:C) into mice. Phagocytosis of RBC-OVA by macrophages and DCs was demonstrated to induce OVA-specific CD4 + and CD8 + T cell activation. Moreover, these CD8 + T cells produced IFN-γ and were able to induce OVA-specific cell lysis. Finally, T-cell response was demonstrated to be dependent on the dose-amount of antigen entrapped and this response could be maintained for up to 30 days.

In vivo circulation of mouse red blood cells frozen in the presence of dextran and glucose

Loading with monosaccharide can improve the quality of human red blood cells (hRBCs) frozen with polymer. But in vivo life span of hRBCs frozen with polymer and sugar is not determined. In this study, following incubation with glucose, mouse red blood cells (mRBCs) were frozen in liquid nitrogen for 24 h using dextran as the extracellular protectant. After thawing, hemolysis, exposure of PS, and osmotic fragility of frozen mRBCs were determined in vitro. After transfusion of fluorescein isothiocyanate (FITC)-labeled mRBCs, the 24 h recovery and half life span of frozen mRBCs were determined. The data indicated the postthaw hemolysis of mRBCs frozen with dextran and glucose were significantly less than that of cells frozen with dextran (17.23% ± 5.21% vs 25.96% ± 10.07%, P = 0.034). But freezing can also result in exposure of phosphatidylserine and increase of osmotic fragility of mRBCs. After transfusion, the 24 h recovery of mRBCs frozen in the absence or presence of glucose was similar to that of the control cells ( P = 0.748 and 0.971). However, the half life span of mRBCs frozen in the absence or presence of glucose was significantly less than that of the control cells ( P = 0.000). In addition, incubation with glucose can not increase the life span of frozen red blood cells (7.16 ± 0.93 d vs 7.15 ± 0.34 d, P = 0.982). In conclusion, incubation with monosaccharide could significantly increase the recovery of mRBCs frozen with polymer. Although freezing can significantly shorten the half life span of frozen cells, it can not influence the 24 h recovery of frozen mRBCs. In addition, incubation with monosaccharide before freezing can not increase the life span of frozen mRBCs. So according to the above data, to increase the life span of hRBCs frozen with polymer and monosaccharide, the osmotic fragility of the frozen RBCs must be decreased in the future.

Implication of oxidative stress as a cause of autoimmune hemolytic anemia in NZB mice

We have recently shown that deficiency of the superoxide dismutase 1 gene (SOD1) causes anemia and autoimmune responses against red blood cells (RBCs) and that transgenic expression of human SOD1 in erythroid cells rescues them. Because these phenotypes observed in SOD1-deficient mice are similar to autoimmune hemolytic anemia (AIHA), a causal connection between reactive oxygen species (ROS) and AIHA was examined using an AIHA-prone New Zealand Black (NZB) mouse. ROS levels in RBCs were high in young NZB mice, compared to control New Zealand White (NZW) mice, and increased during aging. Methemoglobin and lipid peroxidation products were elevated during aging, consistent with the elevated oxidative stress in RBCs of NZB mice. Severity of anemia and levels of intracellular ROS were positively correlated. Levels of antibodies against 4-hydroxynonenal and acrolein were also elevated in NZB mice. Transgenic expression of human SOD1 protein in RBCs of NZB mice suppressed ROS in RBCs and decreased the death rate. When RBCs from C57BL/6 mice were injected weekly into the same strain of mice, production of anti-RBC antibody was observed only in mice that had been injected with oxidized RBCs. Thus, oxidation-mediated autoantibody production may be a more general mechanism for AIHA and related autoimmune diseases.

Design, syntheses and evaluation of hemocompatible pegylated-antimicrobial polymers with well-controlled molecular structures

In this paper, we have designed and synthesized well-defined pegylated-polymers with tertiary amines from readily available commodity monomers 2-(dimethylamino)ethyl methacrylate (DMAEMA) and oligo(ethylene glycol) methyl ether methacrylate (OEGMA, M n ∼ 475 Da) by reversible addition–fragmentation chain transfer (RAFT) polymerisation. By employing a simple and efficient post-polymerisation functionalization strategy, tertiary amines were quaternized to result in cationic polymers. By the careful selection of the functional halide, X–(CH 2) q–R, (where in X = halide; R = the chemical functionality; q = the number of alkyl spacer between the quaternary ammonium group and R), a series of polymers with well-controlled molecular weight, different amphiphilic balance and chemical functionalities (such as alkyl, primary alcohol, primary amine and carboxylic acid) were readily synthesized. The antimicrobial activities of these cationic polymers were determined against Gram-positive bacteria Bacillus subtilis. Minimum inhibitory concentration (MIC), the polymer concentration to completely inhibit the bacterial growth, was found to be dependent both on the nature of functional group and the hydrophobicity of the polymer. Amongst the functional groups, both the alkyl and the alcohol groups were found to be effective, with MIC values in the range of 20–80 mg/L. The haemolytic properties of polymers were analyzed against mouse red blood cells. The poymers with a short alkyl or hydroxyl group demonstrated little haemolysis, yet retained strong antimicrobial activity. The overall hydrophobicity of the polymer influenced its haemolytic behavior. These polymers can be promising antimicrobial agents. In addition, the approach proposed in this study to atom-efficient design and synthesis of antimicrobial polymers from the commercially available monomers can also be applied to develop well-defined functional cationic polymers for various biomedical applications.

Targeting of a Mutant Plasminogen Activator to Circulating Red Blood Cells for Prophylactic Fibrinolysis

Chemical coupling to carrier red blood cells (RBCs) converts tissue type plasminogen activator (tPA) from a problematic therapeutic into a safe agent for thromboprophylaxis. The goal of this study was to develop a more clinically relevant recombinant biotherapeutic by fusing a mutant tPA with a single-chain antibody fragment (scFv) with specificity for glycophorin A (GPA) on mouse RBCs. The fusion construct (anti-GPA scFv/PA) bound specifically to mouse but not human RBCs and activated plasminogen; this led to rapid and stable attachment of up to 30,000 copies of anti-GPA scFv/PA per mouse RBC that were thereby endowed with high fibrinolytic activity. Binding of anti-GPA scFv/PA neither caused RBC aggregation, hemolysis, uptake in capillary-rich lungs or in the reticuloendothelial system nor otherwise altered the circulation of RBCs. Over 40% of labeled anti-GPA scFv/PA injected in mice bound to RBC, which markedly prolonged its intravascular circulation and fibrinolytic activity compared with its nontargeted PA counterpart, anti-GPA scFv/PA, but not its nontargeted PA analog, prevented thrombotic occlusion in FeCl(3) models of vascular injury. These results provide proof-of-principle for the development of a recombinant PA variant that binds to circulating RBC and provides thromboprophylaxis by use of a clinically relevant approach.

A novel acute anemia model for pharmacological research in mice by compelled acute exercise

To generate a novel acute anemia model by compelled exercise. Young adult male mice were compelled to perform an acute exercise by electric stimulation for 30 min. Blood was taken from their tails to be used for detection immediately after exercise. It was found that the red blood cell (RBC) count, hemoglobin concentration and hematocrit of exhausted mice were lower than that of sedentary mice without electric stimulation. The model is in line with the diagnostic criteria. As the mechanisms of the acute anemia model, it was found that the osmotic fragility of the RBC in model mice increased compared to that of normal mice, and that the deformation index, orientation index, small deformation index, deformability and orientation of RBCs in the model mice were reduced. The glutathione (GSH) levels in blood plasma were decreased. The liver, spleen, kidney had no detectable changes. To the best of our knowledge, these effects represent the success of a novel anemia model by compelled acute exercise. The oxidative damages on RBC and hemorheological changes are in part the causes of acute anemia in this model.

Peroxiredoxin-2 (Prx2) Membrane Displacement is A New Additional Factor to the Severity of β-Thalassemic Mouse Red Cell Membrane Damage.

Abstract 2009Poster Board I-1031Peroxiredoxin 2 (Prx2) is the third most abundant cytoplasmic protein in red blood cells (RBCs) and is involved in defence against oxidative stress. Although much is known regarding the structure and function of Prx2 in healthy RBCs, its role in protecting pathological RBCs remains largely unexplored. Here, we studied Prx2 dimerization and membrane translocation in mouse RBCs (wild-type mice, n=20) in response to in vitro oxidative stress by either phenylhydrazine-PHZ (50 μM) or diamide (2mM) or H2O2 (75-100 μM) and in vivo in β thalassemic (β thal) mouse RBCs (Hbbth/th and Hbbth3/+ mouse strains; n=16 from each group).In RBCs lysates we observed (i) slightly increased Prx2 dimerization in PHZ treated RBCs; (ii) complete Prx2 dimerization in presence of diamide; (iii) not apparent Prx2 dimerization in H2O2, suggesting a different sensitivity of Prx2 to the various oxidant agents. In RBCs membrane, Prx2 association was (i) reduced in PHZ treated RBCs; (ii) increased in diamide treated RBCs as both monomers and dimers and (iii) reduced to almost undetectable amount in H2O2 treated RBCs. In order to evaluate whether Prx2 native or from RBCs exposed to oxidative stress possess different degrees of affinity to native or oxidized membrane, we compared Prx2 membrane association in control, diamide, PHZ treated membranes with the cytoplasmic RBC fractions obtained from control, diamide and PHZ treated RBCs. We observed a remarkable loss of interactions only between Prx2 and the membranes obtained from PHZ treated red cells (n=3), suggesting that PHZ may inhibit Prx2 binding to the membrane through the masking of its docking sites. Using multiple techniques (immunofluorescence confocal microscopy, immunoblot and flow cytometric analysis), we showed that Prx2 was displaced from PHZ treated RBC membrane in a dose dependent manner. We then investigated the connection between Prx2 membrane displacement and hemicrome binding in time course experiments with PHZ treated RBCs. Prx2 was displaced from the membrane concomitantly to hemichrome membrane binding, indicating that PHZ blocks the Prx2 binding to the membrane through the masking of its docking sites by hemichromes.Since PHZ membrane oxidative damage mimics that of b thal RBCs we have examined Prx2 in RBCs from two mouse models of b thalassemia. We showed that Prx2 content was higher in b thal mouse RBCs than in controls and correlated with the clinical severity of b thalassemia, but the amount of Prx2 associated to the membrane was markedly reduced as observed in PHZ-treated RBCs. These data suggest that in b thal RBCs the masking of Prx2 membrane binding sites by hemichromes and the accumulation of its oxidized/dimerized state in the cytoplasm may affect Prx2 catalytic efficiency on b thal membrane proteins, therefore contributing to their lack of repair from oxidative insults. Thus, the abnormally low membrane association of Prx2 in b thal mouse RBCs represents a new additional factor amplifying the oxidative damage characterizing b thal RBCs and responsible for their reduced lifespan in the periferal circulation. Disclosures:No relevant conflicts of interest to declare.

Soluble Components of Histoplasma Capsulatum var. Capsulatum have Hemagglutinin Activity and Induce Syngeneic Hemophagocytosis In Vitro

Histoplasma capsulatum var. capsulatum is a thermally dimorphic fungus that causes histoplasmosis. Fungal hemagglutination activity and cases of reactive hemophagocytic syndrome (RHS) have been reported in the disseminated form of disease. In the present study, soluble components of H. capsulatum var. capsulatum have been investigated for hemagglutinin activity and the capacity to induce hemophagocytosis in the mouse system. To analyze hemagglutinating activity, mouse red blood cells (RBC) (1% v/v in PBS) were incubated (37 degrees C, 1 h) with cell-free antigen (CFAg) from H. capsulatum var. capsulatum (isolate IMT/HC128) (RBC-CFAg) or previously heated CFAg (56 degrees C, 30 min) (RBC-hCFAg) or as control with PBS (RBC-PBS). Hemophagocytosis was analyzed by incubating BALB/c mouse peritoneal phagocytic cells (5 x 10(6) cells) with syngeneic RBC, sensitized or not with CFAg. In addition, mouse polyclonal antibodies were raised against syngeneic RBC-CFAg (anti-RBC-CFAg) and used to analyze CFAg chromatographic fractions (Sephadex G75/120) by immunoenzymatic assay (ELISA). Hemagglutinin activity was observed with RBC-CFAg, but not with RBC-hCFAg or RBC. Also, hemophagocytosis was observed with RBC-CFAg, but not with RBC. The anti-RBC-CFAg antibodies reacted with CFAg fractions corresponding to a molecular mass (MM) higher than 150 kDa. In conclusion, the yeast form of H. capsulatum var. capsulatum releases thermolabile soluble components with hemagglutinin activity and it has been demonstrated for the first time that soluble components of the same fungus induce syngeneic hemophagocytosis in the in vitro mouse system. Also, indirect analysis with antibodies suggests that high-MM components (>150 kDa) are responsible for the interaction with RBC.

Rescue of anaemia and autoimmune responses in SOD1-deficient mice by transgenic expression of human SOD1 in erythrocytes

Oxidative stress has been implicated as a cause of various diseases such as anaemia. We found that the SOD1 [Cu,Zn-SOD (superoxide dismutase)] gene deficiency causes anaemia, the production of autoantibodies against RBCs (red blood cells) and renal damage. In the present study, to further understand the role of oxidative stress in the autoimmune response triggered by SOD1 deficiency, we generated mice that had the hSOD1 (human SOD1) transgene under regulation of the GATA-1 promoter, and bred the transgene onto the SOD1(-/-) background (SOD1(-/-);hSOD1(tg/+)). The lifespan of RBCs, levels of intracellular reactive oxygen species, and RBC content in SOD1(-/-);hSOD1(tg/+) mice, were approximately equivalent to those of SOD1(+/+) mice. The production of antibodies against lipid peroxidation products, 4-hydroxy-2-nonenal and acrolein, as well as autoantibodies against RBCs and carbonic anhydrase II were elevated in the SOD1(-/-) mice, but were suppressed in the SOD1(-/-);hSOD1(tg/+) mice. Renal function, as judged by blood urea nitrogen, was improved in the transgenic mice. These results rule out the involvement of a defective immune system in the autoimmune response of SOD1-deficient mice, because SOD1(-/-);hSOD1(tg/+) mice carry the hSOD1 protein only in RBCs. Metabolomic analysis indicated a shift in glucose metabolism to the pentose phosphate pathway and a decrease in the energy charge potential of RBCs in SOD1-deficient mice. We conclude that the increase in reactive oxygen species due to SOD1 deficiency accelerates RBC destruction by affecting carbon metabolism and increasing oxidative modification of lipids and proteins. The resulting oxidation products are antigenic and, consequently, trigger autoantibody production, leading to autoimmune responses.

A novel mouse model of red blood cell storage and posttransfusion in vivo survival

Storage of red blood cells (RBCs) is necessary for an adequate blood supply. However, reports have identified potential negative sequelae of transfusing stored RBCs. An animal model would be useful to investigate the pathophysiology of transfusing stored RBCs. However, it has been reported that storage of rat RBCs in CPDA-1 resulted in an unexpected sudden decline in posttransfusion survival. A mouse model of RBC storage and transfusion was developed to assess survival kinetics of mouse RBCs. RBCs expressing green fluorescent protein were collected in CPDA-1, filter leukoreduced, adjusted to a 75% hematocrit, and stored at 4°C. At weekly intervals, stored RBCs were transfused into C57BL/6 recipients. RBC survival was measured by flow cytometry and chromium-51 labeling. Phosphatidylserine externalization and CD47 expression was also evaluated. Mean 24-hour survivals of transfused RBCs were 99, 91, 64, 54, 30, and 18% after 0, 7, 14, 21, 28, and 35 days of storage, respectively. Stored RBCs showed an initial rapid clearance with subsequent extended survival. Increased surface phosphatidylserine and decreased CD47 expression were also observed. Mouse RBCs showed a progressive decline in survival, as a function of storage time, unlike the precipitous loss of viability reported for rat RBCs. Moreover, changes in the measured surface markers were analogous to trends reported for human RBCs. Together, these findings provide an initial characterization of a novel mouse model of RBC storage with the potential to serve as an experimental platform for studying the pathophysiologic consequences of transfusing stored RBCs.

Changes in red cell ion transport, reduced intratumoral neovascularization, and some mild motor function abnormalities accompany targeted disruption of the Mouse Kell gene (Kel)

Kell (ECE-3), a highly polymorphic blood group glycoprotein, displays more than 30 antigens that produce allo-antibodies and, on red blood cells (RBCs), is complexed through a single disulfide bond with the integral membrane protein, XK. XK is a putative membrane transporter whose absence results in a late onset form of neuromuscular abnormalities known as the McLeod syndrome. Although Kell glycoprotein is known to be an endothelin-3-converting enzyme, the full extent of its physiological function is unknown. To study the functions of Kell glycoprotein, we undertook targeted disruption of the murine Kel gene by homologous recombination. RBCs from Kel(-/-) mice lacked Kell glycoprotein, Kell/XK complex, and endothelin-3-converting enzyme activity and had reduced levels of XK. XK mRNA levels in spleen, brain, and testis were unchanged. In Kel(-/-) mice RBC Gardos channel activity was increased and the normal enhancement by endothelin-3 was blunted. Analysis of the microvessels of tumors produced from LL2 cells indicated that the central portion of tumors from wild-type mice were populated with many mature blood vessels, but that vessels in tumors from Kel(-/-) mice were fewer and smaller. The absence of Kell glycoprotein mildly affected some motor activities identified by foot splay on the drop tests. The targeted disruption of Kel in mouse enabled us to identify phenotypes that would not be easily detected in humans lacking Kell glycoprotein. In this regard, the Kell knockout mouse provides a good animal model for the study of normal and/or pathophysiological functions of Kell glycoprotein.

Bactrocerin‐1: A novel inducible antimicrobial peptide from pupae of oriental fruit flyBactrocera dorsalisHendel

A novel antimicrobial peptide, Bactrocerin-1, was purified and characterized from an immunized dipteran insect, Bactrocera dorsalis. Bactrocerin-1 has 20 amino acid residues with a mass of 2,325.95 Da. The amino acid sequence of Bactrocerin-1 showed very high similarity to the active fragment (46V-65S-NH(2)) of Coleoptericin A. The composition of amino acid residues revealed that Bactrocerin-1 is a hydrophobic, positively charged, and Lys/Ile/Gly-rich peptide. Minimal growth inhibition concentration (MIC) measurements for synthesized Bactrocerin-1 showed a very broad spectrum of anti-microbial activity against Gram-positive bacteria, Gram-negative bacteria, and fungi. Bactrocerin-1 did not show hemolytic activity toward mouse red blood cells even at a concentration of 50 microM. Analysis of the Helical-wheel projection and the CD spectrum suggested that Bactrocerin-1 contains the amphipathic alpha-helix.

Rate of red blood cell destruction varies in different strains of mice infected with Plasmodium berghei-ANKA after chronic exposure

BackgroundSevere malaria anaemia in the semi-immune individuals in the holo-endemic area has been observed to occur at low parasite density with individual variation in the responses. Thus the following has been thought to be involved: auto-immune-mediated mechanisms of uninfected red blood cell destruction, and host genetic factors to explain the differences in individual responses under the same malaria transmission. In this study, the extent of red blood cell (RBC) destruction in different strains of semi-immune mice model at relatively low parasitaemia was studied.MethodologyTo generate semi-immunity, four strains of mice were taken through several cycles of infection and treatment. By means of immunofluorescent assay and ELISA, sera were screened for anti-erythrocyte auto-antibodies, and their relationship with haematological parameters and parasitaemia in the strains of semi-immune mice was investigated.ResultsUpon challenge with Plasmodium berghei ANKA after generating semi-immune status, different mean percentage haemoglobin (Hb) drop was observed in the mice strains (Balb/c = 47.1%; NZW = 30.05%; C57BL/6 = 28.44%; CBA = 25.1%), which occurred on different days for each strain (for Balb/c, mean period = 13.6 days; for C57BL/6, NZW, and CBA mean period = 10.6, 10.8, 10.9 days respectively). Binding of antibody to white ghost RBCs was observed in sera of the four strains of semi-immune mice by immunofluorescence. Mean percentage Hb drop per parasitaemia was highest in Balb/c (73.6), followed by C57BL/6 (8.6), CBA (6.9) and NZW (4.0), p = 0.0005. Consequently, auto-antibodies level to ghost RBC were correlated with degree of anaemia and were highest in Balb/c, when compared with the other strains, p < 0.001.ConclusionThe results presented in this study seem to indicate that anti-RBC auto-antibodies may be involved in the destruction of uninfected RBC in semi-immune mice at relatively low parasite burden. Host genetic factors may also influence the outcome of auto-immune mediated destruction of RBC due to the variation in Hb loss per % parasitaemia and differences in antibody titer for each semi-immune mice strain. However, further studies at the molecular level ought to be carried out to confirm this.

The in vitro antioxidant activity of lotus germ oil from supercritical fluid carbon dioxide extraction

The in vitro antioxidant activity of lotus germ oil extracted by supercritical fluid extraction (SFE) has been investigated. The distinctly high total phenolic compounds content and tocopherol content in lotus germ oil composition were found to be 9.06 ± 0.11% and 485.1 ± 50 mg/100 g, respectively. The lotus germ oil exhibited a dose-dependent inhibitory effect on the hydroxyl free radical and superoxide anion free radical. However, the scavenging effects on the superoxide anion free radical were deceased when the extract concentration was greater than 70 mg/mL. Lotus germ oil showed substantial antioxidant activity in the mice liver and kidney tissues homogenates in a dose-dependent manner. The auto-haemolysis of mice red blood cells was also blocked by lotus germ oil in a dose-dependent manner. Lotus germ oil showed a higher antioxidant activity in the lard system. The high content of phenolic compounds and tocopherol in the lotus germ oil could partially account for the antioxidant activity. These results suggest the lotus germ oil can be used as healthcare oil to develop.

Glutathione Reductase and Thioredoxin Reductase: Novel Antioxidant Enzymes from Plasmodium berghei

Malaria parasites adapt to the oxidative stress during their erythrocytic stages with the help of vital thioredoxin redox system and glutathione redox system. Glutathione reductase and thioredoxin reductase are important enzymes of these redox systems that help parasites to maintain an adequate intracellular redox environment. In the present study, activities of glutathione reductase and thioredoxin reductase were investigated in normal and Plasmodium berghei-infected mice red blood cells and their fractions. Activities of glutathione reductase and thioredoxin reductase in P. berghei-infected host erythrocytes were found to be higher than those in normal host cells. These enzymes were mainly confined to the cytosolic part of cell-free P. berghei. Full characterization and understanding of these enzymes may promise advances in chemotherapy of malaria.

An Assay to Predict L-Asparaginase Neutralization and Enzyme Loaded Red Blood Cells to Maintain in Vivo Efficacy.

E. coli. L-Asparaginase repeated injections induce immunization. Anti-Asparaginase antibodies can provoke clinical hypersensitivity reactions and/or silently inactivate enzyme activity. Consequently, L-Asparaginase clearance is increased, implying a lack of L-asparagine deamination. Firstly, we developed an assay able to detect the presence of neutralizing factors including anti-Asparaginase antibodies. Next we investigated in a mouse model if loading L-Asparaginase into red blood cells (RBC) may be a way to protect its activity against neutralizing factors. A rabbit was immunized injecting 0.5 mg of L-Asparaginase (167 IU) mixed with Freund's adjuvant every 3 weeks for 4-fold. The animal was euthanized and the final serum collected. Part of this final serum was immuno-adsorbed onto protein A for IgG antibodies purification. L-Asparaginase activity was measured by monitoring the kinetics of ammonia generation from the hydrolysis of asparagine. This assay was adapted to a biochemistry automated analyzer. When mixed with undiluted serum from the immunized rabbit, L-Asparaginase activity (0.8 to 100 IU/ml) was totally inhibited for all the concentration range within 15 min at 37°C. In the other hand, up to 1/128 serial dilutions of serum totally inhibited 2 IU/ml L-Asparaginase. As a control, undiluted pre-immunization serum from the same animal did not significantly affect L-Asparaginase activity. To identify the neutralizing factors, IgG from serum were purified by protein-A. As performed with serum, successive dilutions of IgG were mixed with 1.25 IU/ml L-Asparaginase. The IgG inhibited enzyme activity at the 1/128 dilution by 97%, thus proving their neutralizing effect on L-Asparaginase. To simulate the presence of neutralizing antibodies in the patient, we injected 7.5 μg of rabbit IgG into OF1 mice. Control mice were injected with phosphate buffered saline (PBS). Twenty minutes later mice either received 80 IU/kg of native E. coli L-Asparaginase or the same dose entrapped into OF1 mouse RBC. L-Asparaginase was loaded into murine RBC by reversible hypotonic dialysis, followed by a resealing step. The RBC thus acts as a bioreactor where plasmatic asparagine enters and is cleaved by the entrapped L-Asparaginase inside the erythrocyte. L-Asparaginase activity inside the erythrocyte was quantified at 68 IU per ml of erythrocytes, and the extracellular enzyme activity was less to 9% of total enzyme activity. Mice were sacrificed 6 hours after the administration of native or encapsulated L-Asparaginase. Free L-Asparaginase was totally inactivated in plasma of anti-Asparaginase IgG pre-treated mice: 0.002 ±0.002 IU/ml vs 0.417 ±0.103 IU/ml in PBS pre-treated mice. In addition, when L-Asparaginase is loaded inside RBC the activity is maintained irrespective of the presence of antibodies (0.798 ±0.126 IU/ml with IgG vs 0.879 ±0.146 IU/ml without). Moreover asparagine was not deaminated in IgG pre-treated mice who received free L-Asparaginase (27 ±1.6 μmol/L), while below 2 μmol/L in all the other groups. In conclusion, this newly developed assay can predict in vivo L-Asparaginase inefficacy. In addition, L-Asparaginase loaded into RBC is protected against neutralizing antibodies and its efficacy is maintained.

Targeted deletion of α-adducin results in absent β- and γ-adducin, compensated hemolytic anemia, and lethal hydrocephalus in mice

AbstractIn the red blood cell (RBC), adducin is present primarily as tetramers of α- and β-subunits at spectrin-actin junctions, or junctional complexes. Mouse RBCs also contain small amounts of γ-adducin. Platelets contain α- and γ-adducin only. Adducin functions as a barbed-end actin capping protein to regulate actin filament length and recruits spectrin to the ends of actin filaments. To further define adducin's role in vivo, we generated α-adducin knockout mice. α-Adducin is absent in all tissues examined in homozygous null mice. In RBCs, β- and γ-adducin are also absent, indicating that α-adducin is the limiting subunit in tetramer formation at the spectrin-actin junction. Similarly, γ-adducin is absent in α-null platelets. α-Adducin–null mice display compensated hemolytic anemia with features characteristic of RBCs in hereditary spherocytosis (HS), including spherocytes with significant loss of surface area, decreased mean corpuscular volume (MCV), cell dehydration, and increased osmotic fragility. Platelets maintain their normal discoid shape, and bleeding times are normal. α-Adducin–null mice show growth retardation at birth and throughout adulthood. Approximately 50% develop lethal communicating hydrocephalus with striking dilation of the lateral, third, and fourth ventricles. These data indicate that adducin plays a role in RBC membrane stability and in cerebrospinal fluid homeostasis.

Zinc Transporters ZnT1 (Slc30a1), Zip8 (Slc39a8), and Zip10 (Slc39a10) in Mouse Red Blood Cells Are Differentially Regulated during Erythroid Development and by Dietary Zinc Deficiency

Zinc is essential for normal erythroid cell functions and therefore intracellular zinc homeostasis during erythroid differentiation is tightly regulated. However, a characterization of zinc transporters in erythrocytes has not been conducted. The membrane fraction of mature mouse RBC was screened for zinc transporter expression using western analysis as a first step in the characterization process. ZnT1, Zip8, and Zip10 were detected among the 12 transporter proteins tested. We examined expression of these zinc transporters during erythropoietin (EPO)-induced differentiation of splenic erythroid progenitor cells into reticulocytes. Both Zip8 and Zip10 mRNA increased by 2–6 h after addition of EPO to the cells. In contrast, maximal RNA levels for the zinc transporter ZnT1 and erythroid δ-aminolevulinic acid synthase were only produced by 24 h after EPO. We confirmed these changes in transcript abundance by western analysis. Dietary zinc status influences zinc-dependent functions of RBC. To determine whether the identified zinc transporters respond to dietary zinc status, mice were fed a zinc-deficient or control diet. Incorporation of 65Zn into erythrocytes in vitro was significantly increased in cells from the zinc-deficient mice. Western analysis and densitometry revealed that erythrocyte Zip10 was upregulated and ZnT1 was downregulated in the zinc-depleted mice. Zip8 was not affected by restricted zinc intake. Collectively, these data suggest that the zinc transporters ZnT1, Zip8, and Zip10 are important for zinc homeostasis in erythrocytes and that ZnT1 and Zip10 respond to the dietary zinc supply.