Reduced red blood cell destruction by antibody fragments

Antibodies to blood group antigens can cause immune RBC destruction directly (extravascular destruction) or indirectly through subsequent complement activation (intravascular hemolysis). The Fc portion of the IgG antibody is responsible for the effector functions of immune RBC destruction. We hypothesized that sensitization of RBCs with blood group antigen-specific IgG antibodies lacking their Fc portion would escape from the recipient's immune system, allowing for a longer survival period of the RBCs in the circulation. Direct injection of mouse RBC-specific Ter-119 monoclonal antibody into mice resulted in a more severe anemia compared with that in mice injected with the Ter-119 F(ab')2 fragment. We found that mouse RBCs coated in vitro with the Ter-119 F(ab')2 fragment, when transfused into mice, survived longer in circulation compared with RBCs coated with whole Ter-119 IgG molecule. The data support the conclusion that antibodies can be rendered less pathogenic through removal of their Fc portion.     

Evidence for immune-mediated destruction as mechanism for LCMV-induced anemia in persistently infected mice.

A docile substrain of lymphocytic choriomeningitis virus (LCMV) causes a persistent infection in adult C3HeB mice and induces a severe anemia, which, unlike the viremia, eventually resolves. Measurements of red blood cell (RBC) survival rates demonstrated an increased rate of RBC clearance in these animals, indicating a hemolytic process for the anemia. Normal clearance rates of RBCs from infected mice transfused into control mice suggested that there was not an intrinsic defect in these cells. It also appeared that RBC destruction was immune-mediated, as cyclophosphamide treatments prevented the onset of anemia in infected mice, whereas adoptive transfer (AT) of immune splenocytes into immunocompromised mice reestablished the condition. The AT experiments also demonstrated that the onset of anemia correlated with the functional state of the immune cells. In addition, opsonization of RBCs was demonstrated by macrophage phagocytosis, and the appearance of opsonized RBCs corresponded with the course of the anemia. These findings support a hypothesis of RBC opsonization and subsequent phagocytosis by macrophages of the reticuloendothelial system as the mechanism for RBC destruction in LCMV-induced hemolytic anemia.     

An ultrastructural study of the red pulp of the spleen and the liver in unstable hemoglobin hemolytic anemia

Summary Electron microscopic observations on the process of red cell destruction in the spleen and liver of a patient with congenital Heinz body hemolytic anemia, associated with a new variant of unstable hemoglobin, are reported. Two major mechanisms of destruction of Heinz body-containing red cells were noted. One was phagocytosis of these cells in toto by cordal macrophages. The other mechanism, though less significant quantitatively, was intravascular hemolysis of injured red cells in the splenic microvasculature. In the liver, phagocytosis of damaged red cells by Kupffer cells was rare and there was no evidence of intravascular hemolysis in this organ. These morphological findings, together with almost complete recovery from hemolysis following splenectomy, indicated that Heinz body-containing red cells were removed from the circulation predominantly by the spleen. In contrast to experimental Heinz body anemia in animals, Heinz bodies were present even in the nucleated red cells.Supported by a Research Grant for Specific Diseases from the Ministry of Health and Welfare and a Grant-in-Aid for Scientific Research of the Ministry of Education, Japan     

Glutathione and iron at the crossroad of redox metabolism in rats infected by Trypanosoma evansi

The aim of this study was to evaluate the changes in hematological and biochemical parameters of blood during acute Trypanosoma evansi infection in Wistar rats. The end points studied were hematologic parameters, red blood cell fragility, iron content, and glutathione and lipid peroxidation levels. Forty-eight animals were infected with trypomastigotes and distributed into five groups according to the level of parasitemia. Twelve non-inoculated animals were used as control. Parasitemia increased progressively, reaching highest scores at 15 days post-inoculation. At this point, several deleterious effects were observed such as an increase in iron content, in osmotic fragility, and in lipid peroxidation index, while glutathione decreased drastically. These changes were highly correlated to parasitemia (p?<?0.0001) and among each other (p?≤?0.001). Hematological indices (Hb, packed cell volume (PCV), red blood cells (RBC), and mean corpuscular hemoglobin concentration) were also correlated to parasitemia (p?≤?0.0003) but failed to correlate to the other variables. Along with increase in iron, RBC fragility produced a decrease in RBC, PCV, and Hb, but not in mean corpuscular volume. Decrease in glutathione was negatively correlated to the end products of lipid peroxidation, clearly indicating the establishment of a pro-oxidant condition. The results show that the infection causes hematological impairments, increases iron and osmotic fragility, along with marked oxidative stress in red blood cells of rats inoculated with T. evansi.     

Differences between the activities of human monoclonal IgG1 and IgG3 anti-D antibodies of the Rh blood group system in their abilities to mediate effector functions of monocytes.

Seven IgG1 and seven IgG3 human monoclonal antibodies derived from heterohybridoma or Epstein-Barr virus-transformed lymphocytes and specific for the D antigen of the human Rh blood group system were tested for their ability to bring about red cell attachment to and phagocytosis by monocytes. The antibodies produced by the heterohybridomas were also investigated for their potency to mediate antibody-dependent cellular cytotoxicity (ADCC) by monocytes. When red cells were sensitized with any of the IgG1 anti-D antibodies, most of them were ingested by the phagocytes. By contrast, many of the red cells coated with any of the IgG3 antibodies remained attached to the monocyte surface while only few underwent phagocytosis. Some of the attached red cells remained on the phagocyte exterior for a considerable length of time. The ADCC activities of the IgG3 anti-D antibodies was greater than that of the IgG1 anti-D antibodies. The results mean that in vitro IgG1 anti-D mediates red cell destruction mainly by phagocytosis, while IgG3 anti-D causes their destruction predominantly by prolonged cytolysis. These differences between the effector functions of human monoclonal IgG1 and IgG3 anti-D antibodies might have important implications for their use in the prophylaxis of haemolytic disease of the new-born.     

Clinical correlation of positive direct antiglobulin tests in patients with sickle cell disease

Serologic findings of immune-mediated hemolytic anemia (autoimmune hemolytic anemia and cold agglutinin disease) are not infrequent in patients with sickle cell disease and can he clinically significant. Features of sickle cell disease that may affect the emergence and intensity of immune-mediated hemolysis include the antigenic stimulation of chronic red blood cell (RBC) transfusions, increased autoantibody production, RBC membrane defects, and functional asplenism. We describe two patients with sickle cell disease and serologic findings of autoimmune hemolytic anemia, but only one had increased RBC destruction attributed to the autoantibody. That patient's RBCs had IgG and complement on the surface, while those of the other patient had IgG without complement. Functional asplenism may diminish the role of an IgG autoantibody that does not hind complement, since RBCs coated with complement are removed by the liver. Therefore, complement-binding autoantibodies may have particular significance in immune-mediated hemolysis in patients with sickle cell disease.     

Impaired red cell deformability in established adjuvant arthritic disease in rats

Deformablity and fragility of red blood cells (RBC) of established arthritic rats were measured by a filtration technique. They showed a significantly decreased deformability and, accordingly, a considerably increased fragility to micro heat stimulation, induced by laser irradiation.Subacute treatment of arthritic rats with aspirin and indomethacin had no effect on RBC behaviour whereas hydrocortisone restored the deformability of RBC almost to normal. Treatment of RBC of arthritic rats with neuraminidase caused the deformability of the cells to be restored to a supranormal level.     

Haematological changes of cats with Toxoplasma gondii-specific antibodies

The purpose of this study is to access the usefulness of haematological parameters in diagnosing of chronic toxoplasmosis in cats. Antibodies of Toxoplasma gondii were detected in 50 cats using an enzyme-linked immunosorbent assay. Immunoglobulin M (IgM) and immunoglobulin G (IgG) were measured with the cutoff point for T. gondii considered to be 1:64. Fifteen (30%) of the cats were found to be seropositive. No statistically significant difference was found amongst different age groups, but the male cats showed significantly higher antibody titres than female cats. A significant positive correlation was found between IgM and IgG of the cats. Comparison of haematological data between two groups of the cats (IgM <1/64, n = 35; IgM ≥1/64, n = 15) showed that packed cell volume (PCV), red blood cell (RBC) and monocyte values in cats with higher IgM titres were significantly higher than cats with lower IgM. It is suggested that in cases with higher values of PCV, RBC and monocyte in routine complete blood count profile of apparently normal cats, toxoplasmosis should be considered. Further studies with more cases are needed to ensure the diagnostic values of these measured parameters.     

Effect of membrane-bound IgG and desialysation in the interaction of monocytes with senescent erythrocytes

Abstract Determination of the erythrocyte lifespan is a complex process affected by many cellular parameters. In the present study we measured and characterised the red blood cell (RBC) membrane proteins, mainly band 3, and quantified membrane-bound IgG in senescent RBC (SeRBC) and young RBC (YRBC). We also investigated, through a functional assay, the interaction between SeRBC and peripheral blood monocytes. We applied this erythrophagocytosis assay to study the phagocytosis of desialysed RBC. The results obtained showed no changes in the protein content between SeRBC and YRBC and no differences when examining membrane proteins by SDSPAGE. Then, considering that the accumulation of autologous IgG on RBC membrane provides a direct mechanism for the removal of SeRBC, we measured the IgG content of intact RBC using an enzyme-linked antiimmunoglobulin test finding that the number of IgG molecules bound to SeRBC was significantly higher than that observed for YRBC. The increase observed in the percentage of erythrophagocytosis with SeRBC and sensitised RBC (SRBC) confirmed the involvement of autologous IgG in the selective removal of erythrocytes. We also observed a higher percentage of monocytes with phagocytosed and adherent RBC (AM) obtained with neuraminidase-treated RBC than those obtained with YRBC. This finding suggests that a decrease in sialic acid content of SeRBC may be involved in physiological erythrophagocytosis     

Clearance of red cells by monoclonal IgG3 anti-D in vivo is affected by the VF polymorphism of Fcgamma RIIIa (CD16)

Human red cells (RBC) coated with IgG anti-D are cleared from the circulation to the spleen by macrophages which express IgG receptors (Fcgamma R). Polymorphisms of Fcgamma RIIa and Fcgamma RIIIa affect IgG binding in vitro, and may alter the efficiency of clearance of immune complexes in vivo. In a RBC clearance study, 22 Rh D-negative subjects were given 100-400 micro g human monoclonal or polyclonal IgG anti-D i.m. followed 48 h later by 51Cr-labelled D+ RBC. The half lives of the infused D+ RBC were determined, together with the coating levels of anti-D on the D+ RBC. Fcgamma RIIA and FcgammaRIIIA genotyping was performed. Large ranges of phagocytosis and extracellular lysis of RBC in vitro, and of half lives of RBC in vivo, were observed. Clearance of RBC coated with monoclonal IgG3 anti-D (BRAD-3) was more rapid in five subjects homozygous for Fcgamma RIIIa-F/F158 than in three subjects expressing the Fcgamma RIIIa-V158 allele (P = 0.024). This effect was not observed, however, for those individuals given polyclonal anti-D. There was also no significant difference in the efficiency of RBC destruction in vitro or of RBC clearance in vivo between the subjects analysed for individual genotypes or alleles or combinations of alleles. In conclusion, the presence of the Fcgamma RIIIa-V158 allele compromised the efficiency of removal of RBC coated with IgG3 anti-D.     

驻极体对烫伤大鼠心肌损伤和血液流变指标的修复研究

目的:研究驻极体对烫伤大鼠心肌损伤和血液流变学指标的修复作用。方法:将大鼠随机分成对照组、烫伤组和驻极体治疗组,分别测量对照组、烫伤组和驻极体治疗组大鼠血压、全血粘度和红细胞电泳率的变化。结果:大鼠烫伤后的全血粘度较对照组呈显著性提高,红细胞电泳率和颈动脉血压较对照组均有明显下降。负极性驻极体作用烫伤大鼠后,其血液粘度较烫伤组明显下降,而红细胞电泳率和颈动脉血压较烫伤组有明显上升。结论:负极性驻极体不仅对烫伤大鼠的心肌损伤有积极的修复作用,而且能通过修复红细胞的驻极态来改善烫伤大鼠的血液流变学指标。     

Dietary protein and carbohydrate effects on blood parameters related to stress in cat

Adult cats were adapted to hypoglucidic semi-purified diets containing casein or soya as the protein source to study the effects of a 2 hr immobilization period. Body weight of cats fed hypoglucidic diets was significantly decreased. The control casein group showed higher plasma dopamine-beta-hydroxylase activity but lower pyridoxal 5'-phosphate level than control soya group. Cats fed hypoglucidic casein diet, plasma glucose, insulin and pyridoxal 5'-phosphate levels were increased whereas cats fed hypoglucidic soya diet, pyridoxal 5'-phosphate levels were decreased and dopamine-beta-hydroxylase activity increased when data were compared to their respective control groups. A 2 hr immobilization period induced hyperglycemia in all groups whereas cats fed soya diets, plasma insulin level and dopamine-beta-hydroxylase activity were significantly increased and pyridoxal 5'-phosphate content significantly decreased. These results demonstrate that dietary casein and soya protein might be differentiated on a physiological basis and immobilization emphasized the biochemical disturbances observed between the groups thus suggesting a greater resistance to stress in casein groups than in soya groups.     

Hyper-response of serum IgG1 to Staphylococcus aureus peptidoglycan in patients with hyper-IgE syndrome.

The hyper-IgE (HIE) syndrome is characterized by high IgE serum levels, chronic dermatitis and recurrent infections. To determine whether an impairment of the antibody response to Staphylococcus aureus contributes to infections in this syndrome we measured total serum IgG subclass, specific IgG1 and IgG2 levels against peptidoglycan (PG), the immunodominant cell wall component of S. aureus and serum opsonic activity to PG. Of the 14 patients with HIE syndrome, nine had increased level of serum IgG1 and six had IgG2 subclass deficiency. In regard to specific response of IgG1 and IgG2 antibodies to PG, patients were divided into five groups related to ages and compared with 10 control subjects for each age cohort. Patients with HIE syndrome had significant high levels of serum-specific IgG1 to PG and significant decreased levels of serum-specific IgG2 to PG in all five groups. Additionally, serum opsonic activity in patients was significantly higher than that in normal control subjects. It is concluded that IgG2 deficiency or poor IgG2 antibody response to S. aureus is not the explanation of the abnormal susceptibility to S. aureus infections of HIE patients.     

A novel hemoglobin-adenosine-glutathione based blood substitute: evaluation of its effects on human blood ex vivo

Chemically modified hemoglobin (Hb) solutions are under current investigation as potential red cell substitutes. Researchers at Texas Tech University have developed a novel free Hb based blood substitute product. This blood substitute is composed of purified bovine Hb cross-linked intramolecularly with o-adenosine-5'-triphosphate and intermolecularly with o-adenosine, and conjugated with reduced glutathione (GSH). In this study, we compared the effects of our novel blood substitute and unmodified (U) Hb, by using allogenic plasma as the control, on human blood components: red blood cells (RBCs), platelets, monocytes (Mo), and low-density lipoproteins (LDLs). The pro-oxidant potential of both Hb solutions on RBCs was examined by the measurement of osmotic and mechanical fragility, conjugated dienes (CD), lipid hydroperoxides (LOOH), thiobarbituric acid reactants (TBAR-S), isoprostanes (8-iso PGF2alpha) and intracellular GSH. The oxidative modification of LDLs was assessed by CD, LOOH, and TBAR-S, and the degree of apolipoprotein (apo) B cross-linking. The effects of Hb on platelets have been studied by monitoring their responses to the aggregation agonists: collagen, ADP, epinephrine, and arachidonic acid. Monocytes were cultured with Hb solutions or plasma and tested for TNF-alpha and IL-1beta release, then examined by electron microscopy. Results indicate that native UHb initiates oxidative stress of many blood components and aggravates inflammatory responses of Mo. It also caused an increase in RBC osmotic and mechanical fragility (p < 0.001). While the level of GSH was slightly changed, the lipid peroxidation of RBC increased (p < 0.001). UHb was found to be a stimulator of 8-iso PGF2alpha synthesis, a potent modulator of LDLs, and an effective potentiator of agonist induced platelet aggregation. Contrarily, our novel blood substitute did not seem to induce oxidative stress nor to increase Mo inflammatory reactions. The osmotic and mechanical fragility of RBCs was similar to that of the control. Such modified Hb failed to alter LDLs, increase the production of 8-iso PGF2alpha, but markedly inhibited platelet aggregation. The effect of this novel blood substitute can be linked with the cytoprotective and anti-inflammatory properties of adenosine, which is used as a cross-linker and surface modifier, and a modification procedure that lowers the hemoglobin pro-oxidant potential.     

An age-specific cell antigen is present on senescent human red blood cell membranes. A brief note

Immunoglobulin G autoantibodies selectively bind to senescent human red blood cells (RBC) in situ and initiate their removal by phagocytosis. In this paper, we characterize the IgG binding receptor appearing on senescent RBC using glycophorin-enriched vesicles prepared by Triton X-100 extraction of young, middle-aged, and old RBC populations. These vesicles contain all known sialoglycoproteins and trace contaminants of other proteins. IgG binds predominantly to vesicles from old cells, as determined by both ¹²⁵I-labeled protein A binding to IgG molecules and an erythrophagocytosis-inhibition assay. Addition of lipids does not alter IgG binding. Liposomes prepared from lipids of young and old cell fractions do not bind significant amounts of IgG. IgG binding is reduced following trypsin treatment of vesicles. The data suggest that the age-specific cell antigen is a protein which co-purifies with sialoglycoproteins, but is not identical with glycophorin. Since it is extracted predominantly from senescent cells, a chemical modification within the membrane may either form the age-specific cell antigen during aging or render it accessible during senescence.     

Protective effect of severe magnesium deficiency on Plasmodium chabaudi infection

Mice were fed for 30 days on purified diets containing 50 (severely Mg deficient diet), 100 (moderately Mg deficient diet) and 1300 mg/kg (control diet). An additional group raised on stock UAR diet was also used for the experiment. The mice were maintained on the experimental diets for 12 days before being inoculated with P. chabaudi. Infection evolved similarly in mice fed the control purified diet, moderately Mg deficient diet and the stock diet whereas the severely Mg deficient diet induced a 50% decrease in malarial infection as shown by the decrease in the percentage of parasitized red blood cells (RBC). In control mice, RBC Mg values increased significantly during P. chabaudi infection; however RBC Mg values were significantly lower in Mg-deficient than in control animals.     

The ability of normal human monocytes to phagocytose IgG-coated red blood cells is related to the number of accessible galactosyl and mannosyl residues in the Fc domain of the anti-red blood cell IgG antibody molecules

The percentage of normal human monocytes (MCs) that are able to form rosettes with, and subsequently phagocytose, IgG-coated red blood cells (RBCs) has been determined in vitro using five batches of anti-RBC IgG antibodies. These antibodies differed from each other by their capacity to bind to lectins recognizing two of the oligosaccharide structures of the Fc domain, namely, peanut agglutinin (PNA) and concanavalin A (ConA) which specifically bind to beta-galactosyl and alpha-mannosyl residues, respectively. The threshold between high (H) and low (L) binding capacities (BC) was arbitrarily fixed at 15% of mean specific binding. For each level of RBC sensitization tested (1500-6000 Ab molecules/one RBC), the percentage of MCs binding at least three IgG-RBCs was similar whatever the IgG Ab preparations used. In contrast, the percentage of MCs capable of phagocytosing at least three IgG-RBCs coated with 3000, 4500 and 6000 IgG/cell, as well as the phagocytosis index (number of IgG-RBCs ingested/100 MCs) of IgG-RBCs coated with 1500, 3000, 4500 and 6000 IgG/cell, were significantly lower (P less than 0.01 at least) using IgG Ab molecules with either [(PNA-H)(ConA-H)] BC, [(PNA-L) (ConA-H)] BC or with [(PNA-L)(ConA-L)] BC than the corresponding values measured using RBCs coated with IgG Ab molecules exhibiting [(PNA-H)(ConA-L)] BC. The binding to MCs of 125I-labelled anti-RBC IgG Ab molecules exhibiting different binding profiles to PNA and to ConA was studied by Scatchard plot analysis. A single class of binding sites was observed in each case. MCs bound a mean of 23,000 IgG molecules with a mean association constant (Ka) for IgG binding of about 1.4 X 10(8) M-1. These data indicate that terminal (and/or accessible) galactosyl and mannosyl residues of IgG Ab molecules play a role in the ingestion of IgG-RBCs by human MCs, despite the fact IgG Ab binding to IgG(Fc) receptors is not significantly affected. Thus, when studying the phagocytosis of IgG-coated RBC by human MC monolayers, the assay should be performed not only using similar RBC/MC ratios and IgG coating values, but also with IgG antibodies having comparable mean PNA and ConA binding capacities.     

The pathogenesis factors of intraoperative hemolysis in cardiac surgery

As shown in various studies, hemolysis is revealed in all extracorporeal circuits showing the increasing levels of plasma-free hemoglobin (PfHb) during and after cardiopulmonary bypass (CPB). The pathogenesis factors and mechanisms of intraoperative hemolysis are observed in this article. The role of mechanical blood trauma, oxygen free radical generation, activation of complement, preoperative defects erythrocytes, infusion preparations and other medicaments in postperfusion hemolysis are discussed. Along with the complete red blood cell (RBC) destruction (hemolysis), RBCs can also be damaged on a sublethal level, resulting in altered rheological properties, decreased microcirculation and organ dysfunction caused by hypoxia. The severity of the consequences of RBC damage, the high incidence of this complication and the lack of interventional strategies in cases of suspected or confirmed RBC damage are considered, there may be a need for a treatment algorithm for this phenomenon.     

Mechanical fragility calibration of red blood cells

Mechanical fragility of red blood cells (RBCs) is a critical variable for the hemolysis testing of many important clinical devices, such as pumps, valves, and cannulae, and gas exchange devices. Unfortunately, no standardized test for RBC mechanical fragility is currently well accepted. Although many test devices have been proposed for the study of mechanical fragility of RBCs, no one has ever shown that their results have any relevance to a blood pump. Therefore, the fundamental objective of this study was to determine if one or more test devices could be validated as calibrators to document the fragility of the test blood used for any particular test blood. We compared five mechanical fragility test systems to each other and to a Biopump, with respect to hemolysis. All five devices seem to measure the same parameter; the hemoresistometer most closely matched the pump test results, but the stainless steel bead test may be the most practical for routine calibration purposes.     

An approach to measuring RBC haemolysis and profiling RBC mechanical fragility

Red blood cells (RBC) can be damaged by medical products, from storage or from disease. Haemolysis (cell rupture and haemoglobin release) is often a key indicator, with mechanical fragility (MF) offering the potential to assess sub-haemolytic damage as well. This article reports on a unique approach to measuring haemolysis, without the need for centrifugation or other sample separation. It also reports on employing that in measuring blood fragility (susceptibility to haemolysis) under shear stress, utilising an electromagnet to cause a bead to oscillate within a cartridge that contains the sample. Cycling between stressing and optical measurement of induced haemolysis at progressively increasing durations of stress provides a fragility profile. Sub-system-level testing shows high accuracy for the haemolysis measurements and fair consistency for MF profiling. Improving accuracy and precision of profiling is a current focus and a fully integrated and automated version of this system is under development.