Anti-erythrocyte Antibodies Research Articles

Selective Increase of Autoimmune Epitope Expression on Aged Erythrocytes in Mice: Implications in Anti-erythrocyte Autoimmune Responses

We investigated the impact of changes occurring during red blood cell (RBC) ageing on the RBC-binding activity of pathogenic anti-erythrocyte monoclonal antibodies derived from autoimmune-prone New Zealand black (NZB) mice. As assessed by flow cytometric analysis on in vivo biotinylated RBCs, all five NZB-derived anti-RBC mAb exhibited more efficient binding to aged RBCs than to young RBCs, and resulted in a selective elimination of more aged RBCs from the circulating blood. In addition, treatment of RBCs with proteases markedly enhanced the binding of all five anti-RBC mAb, raising the possibility that increased exposure of autoimmune epitopes on aged RBCs may be in part, a result of contacts with proteolytic enzymes during the lifetime of circulating RBCs. In marked contrast, the binding activity of mAb raised in non-autoimmune animals against antigens expressed on RBCs, such as CD44, CD47, CD147 and TER-119, was either decreased or unchanged with RBC ageing, and these epitopes, except for that recognized by anti-CD47 mAb, were highly sensitive to mild treatment with proteases. Our data unravel the unique molecular feature of RBC epitopes involved in autoimmune haemolytic anaemia, suggesting that membrane alterations in aged RBCs might play a significant role in the development of the autoantibody response to RBCs.

Preparation of pure and intact Plasmodium falciparum plasma membrane vesicles and partial characterisation of the plasma membrane ATPase

BackgroundIn host erythrocytes, the malaria parasite must contend with ion and drug transport across three membranes; its own plasma membrane, the parasitophorous membrane and the host plasma membrane. Isolation of pure and intact Plasmodium falciparum plasma membrane would provide a suitable model to elucidate the possible role played by the parasite plasma membrane in ion balance and drug transport.ResultsThis study describes a procedure for isolating parasite plasma membrane from P. falciparum-infected erythrocytes. With this method, the trophozoites released by saponin treatment were cleansed of erythrocyte membranes using anti-erythrocyte antibodies fixed to polystyrene beads. These trophozoites were then biotinylated and the parasite plasma membrane was disrupted by nitrogen cavitation. This process allows the membranes to reform into vesicles. The magnetic streptavidin beads bind specifically to the biotinylated parasite plasma membrane vesicles facilitating their recovery with a magnet. These vesicles can then be easily released from the magnetic beads by treatment with dithiotreithol. The parasite plasma membrane showed optimal ATPase activity at 2 mM ATP and 2 mM Mg2+. It was also found that Ca2+ could not substitute for Mg2+ ATPase activity in parasite plasma membranes whereas activity was completely preserved when Mn2+ was used instead of Mg2+. Other nucleoside triphosphates tested were hydrolysed as efficiently as ATP, while the nucleoside monophosphate AMP was not.ConclusionsWe have described the successful isolation of intact P. falciparum plasma membrane vesicles free of contaminating organelles and determined the experimental conditions for optimum ATPase activity.

Immunoadsorption and Plasma Exchange in Pregnancy

Background: During pregnancy, familial hyperlipidemia or systemic lupus erythematosus (SLE) can exacerbate having devastating consequences for both mother and fetus. Immunoadsorption is established for removal of pathogenic proteins lipoproteins or autoantibodies, but this procedure has only rarely been used in pregnancy. Methods: We evaluated retrospectively 126 extracorporeal treatments during six pregnancies. Forty low-density lipoprotein immunoadsorptions, 6 sole plasma exchanges and 36 combined procedures (plasma exchange followed by immunoadsorption) were performed for severe hypertriglyceridemia, complicated by acute pancreatitis. Forty-four IgG immunoadsorptions were executed in 2 pregnant women suffering from SLE with a disastrous course during prior pregnancies. Results: In hyperlipidemic pregnant women, mean triglyceride levels prior to treatment were 3,841 ± 2,076 mg/dl (mean ± SD) and total cholesterol was 617 ± 354 mg/dl. Until delivery, a 27% reduction of triglycerides could be achieved. Clinical and serological signs of pancreatitis disappeared after initiation of extracorporeal therapy. Four healthy babies were delivered (birthweights between 2,250 and 3,360 g). In 1 woman suffering from SLE, intrauterine fetal death occurred in the 22nd week of gestation despite a reduction of cardiolipin antibodies by 69%. The second case (a twin pregnancy) was complicated by steroid-resistant antibody-mediated anemia. Due to frequent immunoadsorptions, red blood cell count improved (reduction of antierythrocyte antibodies by 66.6%) and 2 healthy babies (birthweights 2,120 and 2,350 g) were delivered by cesarean section. Conclusion: Long-term antibody-based immunoadsorption has been demonstrated to be safe and well tolerated in pregnant women and enables normal intrauterine/fetal development. Although rarely indicated during pregnancy, this treatment modality might be a promising new technique for removal of autoantibodies and lipoproteins in patients with serious gestational complications without sufficient response to conventional therapy.

Aide à la décision en immunohématologie : épreuve directe de comptabilité au laboratoire (edc)

Cross-matching between the serum of a patient and the red blood cells to be transfused is most important for the prevention of hemolytic transfusion reactions in allo-immunized or new-born patients found positive with direct antiglobulin test. Cross-matching is a time-consuming and complex laboratory test. In order to obtain valid results, it is necessary to abide by some technical rules detailed in this article. The choice of the blood units to be cross-matched depends on the patient's clinical story and on the specificity of anti-erythrocyte antibodies present in the serum. The identification and the management of most frequent difficulties met by using the cross-match technique are discussed hereby.

Obstetric uses of intravenous immunoglobulin: Successes, failures, and promises

Intravenous immune globulin (IVIG) is approved for use in a number of conditions that may occur in obstetrical patients, including autoimmune thrombocytopenia and immune deficiency syndromes. IVIG also is widely used in obstetrics for nonapproved indications, such as fetal-neonatal alloimmune thrombocytopenia, antiphospholipid syndrome, and recurrent miscarriage. This review critically analyzes the use of IVIG for these indications based on the best available information. The authors conclude IVIG is effective in the management of fetal-neonatal alloimmune thrombocytopenia. IVIG appears promising as a treatment for severe fetal-neonatal alloimmune hemolysis due to antierythrocyte antibodies. A prospective multicenter trial should be undertaken. IVIG is no more effective than heparin and low-dose aspirin in the treatment of pregnancies complicated by antiphospholipid syndrome but has not been adequately evaluated in refractory cases. Finally, pending convincing studies, IVIG is not effective and should not be used for the management of recurrent miscarriage. (J Allergy Clin Immunol 2001;108:S133-8.)

The immune haemolytic anaemias: a century of exciting progress in understanding.

The immune haemolytic anaemias: a century of exciting progress in understanding.

Antibody-induced anemia in fetal sheep: model for hemolytic disease of the fetus and newborn.

Our purpose was to produce a condition analogous to alloimmune hemolytic disease of the fetus and newborn by infusing antierythrocyte antibodies in fetal sheep. Antierythrocyte antibodies were infused intravascularly into late-gestation ovine fetuses over a 10-day period. Fetal blood was sampled daily for complete blood cell counts, blood gases, iron, erythropoietin (EPO), and electrolyte concentrations. Red cell mass (RCM) and blood volume were determined every other day using indicator dilution techniques. Results were compared with eight similarly aged control animals. Statistical analysis included Student t test, three-factor analysis of variance, and least squares regression. The hematocrit in seven fetal sheep receiving antibody infusion declined significantly by 10.3 +/- 1.7%, whereas it increased in control animals 2.3 +/- 0.6% (P <.001). RCM was reduced by 18.9 +/- 3.2% over the 10-day protocol while increasing 34.1 +/- 4.2% in control animals, representing more than a 50% difference in RCM (P <.001). Fetal EPO was significantly increased with lower hematocrit and lower PO(2) (P <.001). As fetal hematocrit declined below 25%, lactate and reticulocytes also increased (P <.001). Plasma iron concentration was not significantly altered (P =.47). The chronically catheterized fetal sheep is a viable model for studying immunologically induced fetal anemia as hematocrit can be titrated and the fall in RCM and hematocrit are associated with fetal hypoxia and elevated EPO as occurs in the anemic human fetus. Furthermore, there appears to be a threshold degree of anemia required to elicit responses as the fetal EPO, PO(2), and lactate appeared unresponsive until hematocrit fell below 25%.

Unavailability of CD147 leads to selective erythrocyte trapping in the spleen

Adhesive interactions with stromal cells and the extracellular matrix are essential for the differentiation and migration of hematopoietic progenitors. In the erythrocytic lineage, a number of adhesion molecules are expressed in the developing erythrocytes and are thought to play a role in the homing and maturation of erythrocytic progenitors. However, many of these molecules are lost during the final developmental stages leading to mature erythrocytes. One of the adhesion molecules that remains expressed in mature, circulating erythrocytes is CD147. This study shows that blockade of this molecule on the cell surface by treatment with F(ab')(2) fragments of anti-CD147 monoclonal antibody disrupts the circulation of erythrocytes, leading to their selective trapping in the spleen. Consequently, mice develop an anemia, and de novo, erythropoietin-mediated erythropoiesis in the spleen. In contrast, these changes were not seen in mice similarly treated with another antierythrocyte monoclonal antibody with a different specificity. These results suggest that the CD147 expressed on erythrocytes likely plays a critical role in the recirculation of mature erythrocytes from the spleen into the general circulation. (Blood. 2001;97:3984-3988)

Additional Stem Cell Therapy for Graft Failure after Allogeneic Bone Marrow Transplantation

In this study we retrospectively evaluated the effect and outcome of a boost dose of donor stem cells without additional chemotherapy or total body irradiation. Between March 1983 and August 1999, 20 of 788 (2.5%) patients receiving allogeneic bone marrow transplantation (BMT) were treated with an additional boost dose of donor cells. The reasons for the use of the boost treatment were primary graft failure (early rejection; n = 7), secondary graft failure including late rejection (n = 10), refractory pure red cell aplasia caused by the remaining recipient cells producing anti-erythrocyte antibodies (n = 2), and donor lymphocyte infusion induced pancytopenia (n = 1). The patients were aged from 17 to 48 years (median age 31 years). The underlying diseases of the patients were severe aplastic anemia in 12 patients, acute myelogenous leukemia in 3, acute lymphocytic leukemia in 3, and chronic myelogenous leukemia in 2. The donors were human leukocyte antigen-identical siblings in 18 cases, 1 mismatched related donor, and 1 unrelated donor. The cell source was bone marrow in 6 cases and peripheral blood progenitor cells in 14. The median interval between BMT and the boost treatment was 7 weeks (range 1–124). No conditioning regimen was given prior to the boost treatment for 11 patients, while 4 received total nodal irradiation (TNI) plus antithymocyte globulin (ATG), 3 ATG alone, and 2 TNI plus steroid. The median infused booster mononuclear cell dose was 2.55 × 10⁸/kg (range 0.28–37.0). Fifteen (75%) patients achieved a hematological recovery. After the boost treatment, 6 of 20 (30%) patients developed acute graft-versus-host disease (GVHD) ≧ grade II, 3 of whom had had prior GVHD. Five (31.3%) of the evaluable 16 patients developed chronic GVHD. The GVHDs were easily controlled using immunosuppressive agents except in the case of 1 patient. Five patients died after the boost treatment; 2 within 30 days, 2 within 60 days, and 1 after 32 months. The causes of death were: 3 engrafment failures, 1 late rejection, and 1 infection following GVHD. With a median follow-up of 31.5 months (range 6–92), the Kaplan-Meier method estimated that the overall survival rate 1 and 3 years after the boost treatment was 80 and 71%, respectively. The survival of patients with primary graft failure was determined to be significantly lower compared to that of patients with secondary graft failure, using the log rank test (p = 0.0176). Disease category, stem cell source, conditioning prior to a boost treatment, and year of boost treatment did not have an influence on survival. We conclude that the reinfusion of donor stem cells is frequently successful in achieving engraftment with rare occurrence of fatal GVHD. Furthermore, relatively good long-term survival was demonstrated.

Migration and Differentiation of Autoreactive B-1 Cells Induced by Activated γ/δ T Cells in Antierythrocyte Immunoglobulin Transgenic Mice

Using normal and transgenic (Tg) mice, we have shown that peritoneal B-1 cells are activated by administration of cytokines or lipopolysaccharide and migrate to other lymphoid organs where they differentiate into antibody-secreting cells. However, little is known about the process of B-1 cell migration and differentiation in vivo. We developed a mouse line by crossing the antierythrocyte antibody Tg mice (HL mice) with TCR-gamma/delta Tg mice specific for a self-thymus leukemia (TL) antigen in the recombination activating gene (RAG)2(-/-) background. In the presence of the self-antigen, Tg gamma/delta T cells increased in number and manifested activated phenotypes. Peritoneal B-1 cells in these mice migrated into mesenteric lymph nodes and differentiated into autoantibody-secreting cells, resulting in strong autoimmune hemolytic anemia. Furthermore, transfer of RAG2(-/-) x HL bone marrow or peritoneal cells into the peritoneal cavity of RAG2(-/-) x TCR-gamma/delta Tg mice gave rise to donor-derived B-1 cells in mesenteric lymph nodes, and these cells produced the autoantibody. Thus, this study demonstrates that the migration of B-1 cells and differentiation into the antibody-secreting cells can be induced by noncognate T cell help and implies the possibility that gamma/delta T cells may induce B-1 cell differentiation in vivo.

Exacerbation of autoantibody-mediated hemolytic anemia by viral infection.

Strong enhancement of the pathogenicity of an antierythrocyte monoclonal antibody was observed after infection of mice with lactate dehydrogenase-elevating virus. While injection of the antierythrocyte antibody alone induced only moderate anemia, concomitant infection with this virus, which is harmless in most normal mice, led to a dramatic drop in the hematocrit and to death of infected animals. In vitro and in vivo analyses showed a dramatic increase in the ability of macrophages from infected mice to phagocytose antibody-coated erythrocytes. These results indicate that viruses can trigger the onset of autoimmune disease by enhancing the pathogenicity of autoantibodies. They may explain how unrelated viruses could be implicated in the etiology of autoantibody-mediated autoimmune diseases.

SPECTRIN-LIKE PROTEINS IN PLANT NUCLEI

We analysed the presence and localization of spectrin-like proteins in nuclei of various plant tissues, using several anti-erythrocyte spectrin antibodies on isolated pea nuclei and nuclei in cells. Western blots of extracted purified pea nuclei show a cross-reactive pair of bands at 220–240kDa, typical for human erythrocyte spectrin, and a prominent 60kDa band. Immunolocalization by means of confocal laser scanning microscopy reveals spectrin-like proteins in distinct spots equally distributed in the nucleoplasm and over the nuclear periphery, independent of the origin of the anti-spectrin antibodies used. In some nuclei tracks of spectrin-like proteins are also observed. No signal is present in nucleoli. The amount and intensity of signal increases when nuclei were extracted, successively, with detergents, DNase I and RNase A, and high salt, indicating that the spectrin-like protein is associated with the nuclear matrix. The labelling is similar in nuclei of various plant tissues. These data are the first that show the presence and localization of spectrin-like epitopes in plant nuclei, where they may stabilize specific interchromatin domains.

Validation analytique en immunohématologie érythrocytaire

In a transfusional or foeto-maternal context, hemolysis by incompatibility due to anti-erythrocyte antibodies (regular or irregular) remains the most frequent and most serious immunological risk in the receiver. In order to prevent this risk, a number of actions must be taken, such as the realization of the immunohematologic analyses for which the methodological practices have been legislated because of their serious clinical consequences. Several elements play a role in the reliability of the analyses and their results: the selection of the reagents and their validation in the routine technique used; the validation of reception; the controls involved in secondary preparations (e.g., blood cells reagent); and the daily internal controls. All this requires the choice of adapted controls and the management of possible anomalies.

Évolution des méthodes en immunohématologie

The immunogenic nature of erythrocyte polymorphism is in variance with the incompatible transfusion. Indeed, the fixing of an antibody on the corresponding antigen generally condemns the cell concerned with its destruction. Therefore, in order to ensure the immunohemolytic safety of the transfusions, it is necessary to avoid an in vivo encounter between antigens and antibodies, whose feasibility study in vitro is a determining element. Because of the requirement standards of such analyses and the preoccupation with the continuous improvement of transfusion safety, the evolution of the methods used in immunohematology is a constant concern for all those involved in the process. Thus, during the last few years, new technologies have been introduced which aim at improving performance and sometimes implementing alternatives to agglutination. This improvement is not limited to the search for an overall increase in specificity-sensitivity; it also takes into account the capability to detect "the clinically significant" as well as the limitations of human reliability, which justifies the introduction of automation and computerization. The whole of these methodological evolutions associated with that of the performance of reagents, legitimate the need to reconsider the realization of erythrocyte typing and the search for anti-erythrocyte antibodies.

Les champs de l'immunohématologie érythrocytaire

Biological tests and clinical practices are both required for the safety of donors and patients in immunohematology. Therefore, the objectives are to require: 1) biological analysis of anti-erythrocyte antibodies and antigens; 2) compatibility tests; 3) analysis for the biological qualification of blood donations; 4) genotyping; 5) qualitative and quantitative studies on antigens and antibodies; 6) clinical stages prior and after biological tests including systems of information, automation and computerization.

Cytochemical and immunocytochemical methods for electron microscopic detection of glucose-6-phosphate dehydrogenase in brain areas

This paper reports on protocols for the cytochemical and immunocytochemical determination of the glucose-6-phosphate dehydrogenase (G6PD) in brain areas by electron microscopy (EM). The cytochemical assay consists of a pre-embedding staining of small and flat tissue blocks, which were first mildly fixed and then floated in a staining mixture based on the reduction of tetrazolium salts by NADPH. Tissue blocks were then washed, post-fixed in OsO 4, dehydrated through graded ethanol concentrations and embedded in resin. Ultrathin sections were then obtained and observed at the EM. The immunocytochemical technique was performed on completely fixed tissues of perfused animals. After the tissue embedding in resin, ultrathin sections were obtained and treated with a primary anti-erythrocyte G6PD antibody, produced and purified in our laboratory. The immunostaining was performed with secondary gold-conjugated antibody. Gold grains were well evident by EM analysis thus revealing the G6PD protein in the subcellular compartments. These protocols are useful to detect peculiar populations of neurons which express high levels of G6PD to sustain processes of neural plasticity in some brain areas. Themes: Development and regeneration Topics: Cell division and differentiation

Expression levels of B cell surface immunoglobulin regulate efficiency of allelic exclusion and size of autoreactive B-1 cell compartment.

Surface-expressed immunoglobulin (Ig) has been shown to have a critical role in allelic exclusion of Ig heavy (H) and light (L) chains. Although various degrees of suppression of endogenous Ig expression are observed in Ig transgenic (Tg) mice, it was not clear whether this difference is due to different onsets of Tg expression or to different levels of Tg expression, which are obviously affected by integration sites of the transgene. In this study we generated antierythrocyte antibody Tg mice that carry tandem joined H and L chain transgenes (H+L) and confirmed that homozygosity of the transgene loci enhances the level of transgene expression as compared with heterozygosity. Suppression of endogenous H and L chain gene expression was stronger in homozygous than in heterozygous Tg mice. Similar results were obtained in control Tg mice carrying the H chain only. These results suggest that there is a threshold of the B cell receptor expression level that induces allelic exclusion. In addition, despite the same B cell receptor specificity, the size of Tg autoreactive B-1 cell compartment in the peritoneal cavity is larger in homozygous than in heterozygous mice, although the number of the Tg B-2 cell subset decreased in the spleen and bone marrow of homozygous Tg mice as compared with heterozygous Tg mice. By contrast, homozygosity of the H chain alone Tg line, which does not recognize self-antigens, did not increase the size of the peritoneal B-1 subset. These results suggest that the size of the B-1 cell subset in the Tg mice may depend on strength of signals through B cell receptors triggered by self-antigens.

Antierythrocyte Antiboldies in Hemodialysis and Kidney Transplant Patients

Natural antierythrocytic antibodies may be stimulated by bacterial antigens and the immune type may occur as a result of pregnancy or blood transfusions. The prevalence increases with the number of red cell units transfused. Specificity, on the other hand, depends on ethnical backgrounds. The clinical importance of these antibodies is to precipitate hemolytic transfusion reactions and erythroblastosis fetalis. Hemodialysis patients are multitransfused and have a quite variable prevalence of antibodies. Kidney transplant patients with blood group identity do not form antibodies. We studied the presence of both types of antierythrocytic antibodies (natural and immune) in hemodialysis and kidney transplant patients in Brazilian blood transfusion and nephrology services.

Booster marrow or blood cells for graft failure after allogeneic bone marrow transplantation.

Twenty allogeneic bone marrow transplant patients were treated with an additional dose of donor cells (boost dose) for graft failure (n = 7), partial graft failure (n = 11) or extensive hemolysis caused by remaining recipient cells producing anti-erythrocyte antibodies (n = 2). Donors were in 12 cases HLA-identical siblings, three mismatched related donors and five unrelated donors. Cell source was in 13 cases bone marrow and in seven peripheral blood progenitor cells. Median time from BMT to booster dose was 3.4 months (range 0.7-59.3). Median infused cell dose was 2.4 x 10(8)/kg patient (range 0.5-19.0). As GVHD prophylaxis most patients were already receiving different combinations of cyclosporine, prednisolone and methotrexate. No preparative treatment was given prior to boost in 16 patients; four received ATG. After boost, 11 patients developed acute GVHD, six grade I, four grade II and one grade III. Except for one patient, acute GVHD after boost was less, or the same grade as after BMT. Six patients developed chronic GVHD, three limited and three extensive. Five patients died within 30 days of the boost. Nine of 15 (60%) evaluable patients became transfusion independent within 30 days and three more within 60 days. Causes of death were: infections six (IP four, pneumonia two), relapse three; and GVHD three. Three out of five patients transplanted with unrelated marrow suffered from severe immunological reactions and died 2-3 months after the boost dose. Patient survival 1 and 3 years after boost was 55% and 43%, respectively. Among patients with hematological malignancies, leukemia-free survival at 3 years was 41%.

Silicone Gel Enhances the Development of Autoimmune Disease in New Zealand Black Mice but Fails To Induce It in BALB/cAnPt Mice

Anecdotal evidence links silicone gel breast implants with the development of autoimmune connective tissue disease in women. To investigate whether silicone gel is capable of directly inducing and/or enhancing the development of autoimmune disease, female BALB/cAnPt (BALB/c) and New Zealand Black (NZB) mice were injected subcutaneously with silicone gel, pristane, a nonmetabolizable substance that can cause plasmacytomas in BALB/c and NZB mice, or saline and monitored for the development of glomerulonephritis and autoantibody production. NZB, but not BALB/c, mice spontaneously develop autoantibodies and an autoimmune hemolytic anemia by 12 months of age. Over a period of 10 months, biweekly screening for proteinuria revealed increases in urinary protein in NZB mice that received multiple injections of either silicone gel or pristane. In contrast, urinary protein was unaffected in identically treated BALB/c mice. Although, silicone gel had no effect on serum titers of anti-erythrocyte antibodies in NZB mice, the hematocrits were significantly decreased. Moreover, silicone gel both increased the concentration of IgM anti-type I collagen antibodies and skewed the immunofluorescent staining pattern of serum autoantibodies on HEp-2 cells. In contrast, silicone gel failed to induce the production of anti-erythrocyte or antinuclear antibodies in BALB/c mice and induced only slight increases in IgG anti-type I collagen antibodies. These results suggest that silicone gel can exacerbate the development of autoimmune disease in autoimmune NZB mice, but fails to induce disease in normal BALB/c mice. This is consistent with several epidemiological studies failing to demonstrate an increase in the incidence of autoimmune disease in women with breast implants. However, because silicone gel was able to exacerbate autoimmune disease in NZB mice, it may play a similar role in the development of autoimmune disease in a small percentage of women who are genetically susceptible to such diseases.