Ectopic expression of band 3 anion transport protein in colorectal cancer revealed in an autoimmune hemolytic anemia patient.

Cancer patients occasionally have anemia with high mean corpuscular volume in addition to iron deficiency anemia. Secondary autoimmune hemolytic anemia (AIHA) following cancer is also observed with low frequency. To date, no causal mechanisms for these disease states have been reported. Here, we present the case of an 80-year-old woman with AIHA that was resistant to prednisolone. Further examinations revealed primary adenocarcinoma of the sigmoid colon and primary squamous cell carcinoma in the right lung. After resections of these tumors, her anemia partially improved until a colon cancer-derived metastatic tumor was detected in the left lung. Immunoprecipitation of erythrocyte membrane proteins with an autoantibody followed by mass spectrometry/Western blotting identified band 3 as the target of the autoantibody. Immunohistochemical analysis revealed ectopic expression of band 3 in the colon adenocarcinoma. To our knowledge, this is the first report that identifies the cause in a case of anemia without bleeding in a cancer patient and that defines a mechanism underlying secondary AIHA following cancer progression.

Anti-band 3 and anti-spectrin antibodies are increased in Plasmodium vivax infection and are associated with anemia.

Clearance of non-infected red blood cells (nRBCs) is one of the main components of anemia associated with Plasmodium vivax malaria. Recently, we have shown that anemic patients with P. vivax infection had elevated levels of anti-RBCs antibodies, which could enhance in vitro phagocytosis of nRBCs and decrease their deformability. Using immunoproteomics, here we characterized erythrocytic antigens that are differentially recognized by autoantibodies from anemic and non-anemic patients with acute vivax malaria. Protein spots exclusively recognized by anemic P. vivax-infected patients were identified by mass spectrometry revealing band 3 and spectrin as the main targets. To confirm this finding, antibody responses against these specific proteins were assessed by ELISA. In addition, an inverse association between hemoglobin and anti-band 3 or anti-spectrin antibodies levels was found. Anemic patients had higher levels of IgG against both band 3 and spectrin than the non-anemic ones. To determine if these autoantibodies were elicited because of molecular mimicry, we used in silico analysis and identified P. vivax proteins that share homology with human RBC proteins such as spectrin, suggesting that infection drives autoimmune responses. These findings suggest that band 3 and spectrin are potential targets of autoantibodies that may be relevant for P. vivax malaria-associated anemia.

Frecuency of antigens and alloantibodies of Diego system in blood / Frecuencia del antígeno y aloanticuerpos del sistema Diego en donantes de sangre.

Introduction: The Diego blood group is an irregular blood system which has been involved in cases of hemolytic disease of the newborn and post transfusion reactions, in this system have been identified 22 erythrocyte antigens, among which the pair Dia/Dib is the most important because those have the most immunogenic potential. Objective: This research aims to determine the frequency of antigen Dia and their respective alloantibody in the Ecuadorian population. Methods: It was performed a simple random sampling in the donor population, being later tested tube agglutination by the presence or absence of the antigen and its alloantibody Dia applying gel agglutination technique. Results: It was observed an antigen prevalence of 25% against a 6.09% of percentage alloimmunization due to Dia antigen, without significant differences between men and women and these being independent of the age and origin of the donor, showing that there are some Diego positive cases in Ecuadorian population as probably cases of transfusional alloinmunization or due to fetal-maternal alloinmunization. Conclusions and Recommendations: The frequency distribution of antigens and alloantibodies from the Diego blood group is almost uniform in the population, due presumably to the high incidence of miscegenation in our country. Therefore it becomes vitally important the implementation of this blood system inside the protocols of irregular antibodies identification in Ecuadorian blood banks.

Introducción: El sistema Diego es un sistema sanguíneo irregular involucrado de manera clínica en casos de enfermedad hemolítica del recién nacido y en reacciones postransfusionales, dentro de este sistema de han identificado a 22 antígenos eritrocitarios de los cuales el par Dia/Dib son los de mayor importancia por su potencial inmunogénico. Objetivo: Determinar la frecuencia del antígeno Dia y la identificación del aloanticuerpo en la población ecuatoriana. Métodos: Se realizó un muestreo aleatorio simple y se testó mediante aglutinación en tubo la presencia o ausencia del antígeno y en metodología en gel la presencia del aloanticuerpo anti-Dia. Resultados: Se estableció una prevalencia del antígeno Dia del 25% frente a un 6.09% de aloinmunización por dicho antígeno en donantes de sangre ecuatorianos; no existieron diferencias significativas en la asociación de las variables, siendo estas independientes de la edad y procedencia del donante, sin embargo se pudo constatar que en Ecuador existe población portadora del antígeno Diego Dia y casos de aloinmunización por este sistema debidos probablemente a la incompatibilidad transfusional sea esta por vía maternofetal o por transfusiones de sangre. Conclusiones y recomendaciones: La distribución de la frecuencia de antígenos y aloanticuerpos del sistema Diego es casi uniforme dentro de la población de nuestro país, probablemente por ser un territorio con alto grado de mestizaje, por lo que es de vital importancia la implementación de la detección de este sistema sanguíneo y su inclusión dentro de los protocolos de detección de anticuerpos irregulares en los bancos de sangre de Ecuador.

Biocompatible coupling of therapeutic fusion proteins to human erythrocytes.

Carriage of drugs by red blood cells (RBCs) modulates pharmacokinetics, pharmacodynamics, and immunogenicity. However, optimal targets for attaching therapeutics to human RBCs and adverse effects have not been studied. We engineered nonhuman-primate single-chain antibody fragments (scFvs) directed to human RBCs and fused scFvs with human thrombomodulin (hTM) as a representative biotherapeutic cargo (hTM-scFv). Binding fusions to RBCs on band 3/glycophorin A (GPA; Wright b [Wrb] epitope) and RhCE (Rh17/Hr0 epitope) similarly endowed RBCs with hTM activity, but differed in their effects on RBC physiology. scFv and hTM-scFv targeted to band 3/GPA increased membrane rigidity and sensitized RBCs to hemolysis induced by mechanical stress, while reducing sensitivity to hypo-osmotic hemolysis. Similar properties were seen for other ligands bound to GPA and band 3 on human and murine RBCs. In contrast, binding of scFv or hTM-scFv to RhCE did not alter deformability or sensitivity to mechanical and osmotic stress at similar copy numbers bound per RBCs. Contrasting responses were also seen for immunoglobulin G antibodies against band 3, GPA, and RhCE. RBC-bound hTM-scFv generated activated protein C (APC) in the presence of thrombin, but RhCE-targeted hTM-scFv demonstrated greater APC generation per bound copy. Both Wrb- and RhCE-targeted fusion proteins inhibited fibrin deposition induced by tumor necrosis factor-α in an endothelialized microfluidic model using human whole blood. RhCE-bound hTM-scFv more effectively reduced platelet and leukocyte adhesion, whereas anti-Wrb scFv appeared to promote platelet adhesion. These data provide a translational framework for the development of engineered affinity ligands to safely couple therapeutics to human RBCs.

Exacerbation of oxidative stress during sickle vaso-occlusive crisis is associated with decreased anti-band 3 autoantibodies rate and increased red blood cell-derived microparticle level: a prospective study.

Painful vaso-occlusive crisis, a hallmark of sickle cell anaemia, results from complex, incompletely understood mechanisms. Red blood cell (RBC) damage caused by continuous endogenous and exogenous oxidative stress may precipitate the occurrence of vaso-occlusive crises. In order to gain insight into the relevance of oxidative stress in vaso-occlusive crisis occurrence, we prospectively compared the expression levels of various oxidative markers in 32 adults with sickle cell anaemia during vaso-occlusive crisis and steady-state conditions. Compared to steady-state condition, plasma levels of free haem, advanced oxidation protein products and myeloperoxidase, RBC caspase-3 activity, as well as the concentrations of total, neutrophil- and RBC-derived microparticles were increased during vaso-occlusive crises, whereas the reduced glutathione content was decreased in RBCs. In addition, natural anti-band 3 autoantibodies levels decreased during crisis and were negatively correlated with the rise in plasma advanced oxidation protein products and RBC caspase-3 activity. These data showed an exacerbation of the oxidative stress during vaso-occlusive crises in sickle cell anaemia patients and strongly suggest that the higher concentration of harmful circulating RBC-derived microparticles and the reduced anti-band 3 autoantibodies levels may be both related to the recruitment of oxidized band 3 into membrane aggregates.

Host-parasite interaction: multiple sites in the Plasmodium vivax tryptophan-rich antigen PvTRAg38 interact with the erythrocyte receptor band 3.

Tryptophan-rich antigens of malarial parasites interact with host molecules and play an important role in parasite survival. Merozoite expressed Plasmodium vivax tryptophan-rich antigen PvTRAg38 binds to human erythrocytes and facilitates parasite growth in a heterlologous Plasmodium falciparum culture system. Recently, we identified band 3 in human erythrocytes as one of its receptors, although the receptor-ligand binding mechanisms remain unknown. In the present study, using synthetic mutated peptides of PvTRAg38, we show that multiple amino acid residues of its 12 amino acid domain (KWVQWKNDKIRS) at position 197-208 interact with three different ectodomains of band 3 receptor on human erythrocytes. Our findings may help in the design of new therapeutic approaches for malaria.

Identification of sheep red blood cell (SRBC) surface immune-responsive peptides detected by antisera from SRBC-immunized rats.

To identify the sheep red blood cell (SRBC) surface immune-responsive peptides, immuno-reactive fraction of SRBC was detected by SDS-PAGE and western blot analysis with antisera from SRBC-immunized rats. Then the most intense immuno-reactive band on SDS-PAGE was subjected to nanoLC-ESI-MS/MS analysis, and 17 proteins were identified including membrane proteins of erythrocytes such as band 3 anion transport protein isoform 1 (Anion exchange protein 1; AE-1, CD233), Ammonium transporter Rh type A (Rh type A glycoprotein, CD241) and Ankyrin-1 (ANK-1), Spectrin beta chain. Among them, plasma protein AE-1 (CD233) and Rh type A glycoprotein (CD241) have transmembrane domain and correspond to extracellular region in their sequences. These extracellular regions of the plasma membrane proteins are supposed to be major immune-responsive peptides of SRBC in rats. These peptides are promising for the construction of an ELISA system which does not require the processing of SRBC membrane ghosts.

[Molecular mechanisms of autoimmune hemolytic anemia].

Autoimmune hemolytic anemia (AIHA) is an acquired immunological disease in which red blood cells (RBCs) are selectively attacked and destroyed (hemolyzed) by autoantibodies produced by the patient's own immune system. Several hypotheses regarding the mechanisms underlying the development of AIHA have been proposed, but the actual pathogenesis remains unclear. Since the major autoantigens in warm AIHA were determined to be Rh protein, band 3 and glycophorin A in 1993, helper T cells (Th1, Th2 and Th17) and regulatory T (Treg) cells specifically reacting to Rh peptides were reported in patients with AIHA. Recently, Th1 responses were found to be suppressed with synthetic peptides that are recognized by the Treg cells, and Th17 cells and interleukin 17 were shown to contribute to the induction and the development of AIHA. This approach to understanding AIHA pathogenesis may provide clues to finding novel targets for immunotherapy against AIHA.

Interaction of Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 with Band 3 on Human Erythrocyte Surface Facilitates Parasite Growth.

Plasmodium tryptophan-rich proteins are involved in host-parasite interaction and thus potential drug/vaccine targets. Recently, we have described several P. vivax tryptophan-rich antigens (PvTRAgs), including merozoite expressed PvTRAg38, from this noncultivable human malaria parasite. PvTRAg38 is highly immunogenic in humans and binds to host erythrocytes, and this binding is inhibited by the patient sera. This binding is also affected if host erythrocytes were pretreated with chymotrypsin. Here, Band 3 has been identified as the chymotrypsin-sensitive erythrocyte receptor for this parasite protein. Interaction of PvTRAg38 with Band 3 has been mapped to its three different ectodomains (loops 1, 3, and 6) exposed at the surface of the erythrocyte. The binding region of PvTRAg38 to Band3 has been mapped to its sequence, KWVQWKNDKIRSWLSSEW, present at amino acid positions 197-214. The recombinant PvTRAg38 was able to inhibit the parasite growth in in vitro Plasmodium falciparum culture probably by competing with the ligand(s) of this heterologous parasite for the erythrocyte Band 3 receptor. In conclusion, the host-parasite interaction at the molecular level is much more complicated than known so far and should be considered during the development of anti-malarial therapeutics.

Lethal autoimmune hemagglutination due to an immunoglobulin A autoagglutinin with Band 3 specificity.

BACKGROUND: We describe a patient with a high-titer warm immunoglobulin (Ig)A autoantibody resulting in death due to hemagglutination rather than to hemolysis. CASE REPORT: A 47-year-old male patient presented with an intriguing pronounced vascular erythema of the skin. A livedo reticularis associated with cold agglutinin of high thermal amplitude was suspected. The patient's condition unexpectedly and abruptly deteriorated resulting in death 3 days after admission. STUDY DESIGN AND METHODS: Conventional serologic procedures and immunochemical methods were used. RESULTS: Serologic and immunochemical examinations revealed a warm IgA autoantibody of high titer with anti-Band 3 specificity. Although the patient presented with severe anemia, only mild signs of hemolysis were observed, with no evidence of complement activation. The autopsy revealed an enormous accumulation of agglutinated red blood cells in liver and spleen and a B-cell lymphoma and cerebral edema. Thus, the patient's death was largely caused by hypoxia related to hemagglutination rather than to hemolysis and/or anemia per se. CONCLUSION: Strongly hemagglutinating antibodies may not only cause immune hemolysis but also hypoxia due to intravascular hemagglutination.

Membrane peroxidation and methemoglobin formation are both necessary for band 3 clustering: mechanistic insights into human erythrocyte senescence.

Oxidative damage and clustering of band 3 in the membrane have been implicated in the removal of senescent human erythrocytes from the circulation at the end of their 120 day life span. However, the biochemical and mechanistic events leading to band 3 cluster formation have yet to be fully defined. Here we show that while neither membrane peroxidation nor methemoglobin (MetHb) formation on their own can induce band 3 clustering in the human erythrocytes, they can do so when acting in combination. We further show that binding of MetHb to the cytoplasmic domain of band 3 in peroxidized, but not in untreated, erythrocyte membranes induces cluster formation. Age-fractionated populations of erythrocytes from normal human blood, obtained by a density gradient procedure, have allowed us to examine a subpopulation, highly enriched in senescent cells. We have found that band 3 clustering is a feature of only this small fraction, amounting to ∼0.1% of total circulating erythrocytes. These senescent cells are characterized by an increased proportion of MetHb as a result of reduced nicotinamide adenine dinucleotide-dependent reductase activity and accumulated oxidative membrane damage. These findings have allowed us to establish that the combined effects of membrane peroxidation and MetHb formation are necessary for band 3 clustering, and this is a very late event in erythrocyte life. A plausible mechanism for the combined effects of membrane peroxidation and MetHb is proposed, involving high-affinity cooperative binding of MetHb to the cytoplasmic domain of oxidized band 3, probably because of its carbonylation, rather than other forms of oxidative damage. This modification leads to dissociation of ankyrin from band 3, allowing the tetrameric MetHb to cross-link the resulting freely diffusible band 3 dimers, with formation of clusters.

Physiologic autoantibody and immunoglobulin interventions during aging.

Physiologic autoantibodies, that is, those with an active physiologic role, are an important part of the normal human immune system and are essential in maintaining homeostasis. Evidence suggests that the body uses autoantibodies to prevent disease and to self-treat diseases once started. This suggests a potential therapeutic role for autoantibodies, or, even better, a way to use them to prevent disease. Their capacity to remove aged, damaged cells is well established. Immunoglobulin (Ig) G autoantibodies bind to senescent cell antigen (SCA), which is an altered band 3 anion exchanger protein found mainly on aged cells. Once bound, IgG triggers the removal of the senescent cells by macrophages. Band 3 is altered primarily by oxidation, which in turn generates SCA. These studies demonstrated that oxidation can generate neoantigens that the immune system will recognize. Band 3 isoforms are ubiquitous: they have been found in all mammalian cells and species so far examined. The innate immune response to band 3 membrane proteins, and their regulation of cellular lifespan and therapeutic potential will be presented. Examples of other potential innate and physiologic autoantibodies include neuroprotective antibodies to amyloidgenic toxic peptides and antibodies to oxidized LDL (OxLDL), which modify the natural progression of atherosclerosis.

Natural antiband 3 antibodies in patients with sickle cell disease.

Band 3 oligomers, precociously formed in the membrane of sickle red blood cells (SS RBC) as a result of oxidative damage, induce two significant changes: (1) contribution to the adhesive nature of these cells to endothelial cells; (2) production of recognition sites for natural antiband 3 antibodies (antiband 3 Nabs). The inhibition of the adhesion of SS RBC to endothelial cells by band 3 peptides suggests a participation of antiband 3 Nabs in the etiology and prevention of vaso-occlusive crises (VOC). To address this question, we measured the levels of antiband 3 Nabs in sickle cell anaemia (SCA) patients (45 in steady state, 35 in VOC) and in controls (27 sickle trait, 30 normal AA subjects). A significant decreased of antiband 3 Nabs in the VOC group was demonstrated as compared with the steady state group, the sickle trait and healthy controls. This study provides data suggesting that Antiband 3 Nabs are likely to play a role in the SCA VOC.

Living-related liver transplantation in Diego blood group disparity: a case report.

To date, only limited cases of Diego blood group disparity in liver transplantation have been reported, and no cases with a long-term clinical course have been documented. Herein, we report a case of Diego blood group disparity in liver transplantation with details of long-term follow-up. The recipient was a 47-year-old woman with primary biliary cirrhosis; her 18-year-old daughter was the donor. Both recipient and donor were of blood type O according to the ABO blood group system. Preoperative serological tests showed the presence of antibodies against the Di(a) antigen only in the recipient, and not in the donor. Thus, the Diego phenotype was Di(a+) in the donor and Di(a-) in the recipient. Living-related liver transplantation was performed in July 2009. Immediate graft function was obtained, and no signs of humoral or cellular rejection were observed during the postoperative period. Further, anti-Di(a) antibodies were not detected throughout the postoperative course. The patient is alive and shows no signs of humoral rejection 34 months after liver transplantation. Liver transplantation has been performed successfully in cases of Diego blood group disparity.

Naturally occurring autoantibodies in mediating clearance of senescent red blood cells.

Germline-encoded naturally occurring autoantibodies (NAbs) developed about 400 to 450 million years ago to provide specificity for clearance ofbody waste in animals with 3 germ layers. Such NAbs became a necessity to selectively clear aged red blood cells (RBC) surviving 60 to 120 d in higher vertebrates. IgG NAbs to senescent RBC are directed to the most abundant integral membrane protein, the anion-transport protein or band 3 protein, but only bind firmly upon its oligomerization, which facilitates bivalent binding. The main constituent of RBC, the oxygen-carrying hemoglobin, is susceptible to oxidative damage. Oxidized hemoglobin forms hemichromes (a form of aggregates) that bind to the cytoplasmic portion of band 3 protein, induces their clustering on the cytoplasmic, as well as the exoplasmic side and thereby provides the prerequisites for the low affinity IgG anti-band 3 NAbs to bind bivalently. Bound anti-band 3 NAbs overcome their low numbers per RBC by stimulating complement amplification. An affinity for C3 outside the antigen binding region is responsible for a preferential formation of C3b(2)-IgG complexes from anti-band 3 NAbs. These complexes first bind oligomeric properdin, which enhances their affinity for factor B in assembling an alternative C3 convertase.

Identification of a cross-reacting, monoclonal anti-human CD233 antibody for identification and sorting of rhesus macaque erythrocytes.

Erythroid biology research involving rhesus macaques has been applied to several topics including malaria, hemoglobinopathy and gene therapy research. However, analyses of the rhesus red blood cells are limited by the inability to identify and sort those cells in research blood samples using flow cytometry. Here it is reported that the BRIC 6 hybridoma clone raised to the human erythroid surface molecule (referred to as CD233, Band 3, AE1, or SLC4A1) produces cross-reactive and erythroid-specific antibodies for flow cytometric detection and sorting of rhesus macaque erythrocytes.

A new cold autoagglutinin specificity: the third external loop of band 3.

BACKGROUND: Cold autoagglutinins (CAs) are almost always of immunoglobulin (Ig)M class and specific for carbohydrate antigens. We report a case of hemolytic anemia caused by a mixture of IgM and IgG cold agglutinins. Both agglutinins were specific for the third extracellular loop of band 3. CASE REPORT: The patient, a 46-year-old man with no medical history, was admitted for an acute, life-threatening hemolytic anemia caused by a cold agglutinin. Improvement was obtained after plasmapheresis, red blood cell (RBC) transfusions, and steroid therapy. Recovery was complete and no etiology was found. STUDY DESIGN AND METHODS: Conventional serologic procedures and immunochemical methods were used. RESULTS: The patient's autoantibody was directed to an antigen equally expressed by adult and cord RBCs, resistant to papain, neuraminidase, and endo-beta-galactosidase treatments. This pattern of reactivity excluded all known specificities of cold agglutinins. The antibody failed to react with RBCs treated by alpha-chymotrypsin or pronase, suggesting that it may be directed to the third extracellular loop of band 3. Serum fractionation showed that the cold agglutinin was composed of IgM and IgG class antibodies. Antibody specificity was confirmed by blocking tests using murine monoclonal antibodies against band 3 and by immunoprecipitation. CONCLUSION: This is to our knowledge the first observation of a CA specific for band 3. The coexistence of IgM and IgG molecules, a very unusual feature for a CA, may be related to the peptidic nature of the target antigen.

Naturally occurring anti-band 3 antibodies and red blood cell removal under physiological and pathological conditions.

Naturally occurring antibodies (NAbs) directed to band 3 protein (major erythrocyte membrane protein) are involved in the clearance of red blood cell (RBC) at the end of their lifespan as well as in the removal of RBC in different hereditary haemolytic disorders and in malaria. In all cited situations RBC undergoes oxidative stress and hemichromes (haemoglobin degradation products) are formed. Hemichromes possess a strong affinity for band 3 cytoplasmic domain and, following their binding, lead to band 3 oxidation and clusterisation. Those band 3 clusters show increased affinity for NAbs which activate complement and finally trigger the phagocytosis of altered RBC. During intra-erythrocytic malaria parasite growth, NAbs begin to bind to RBC surface at early parasite development stages increasing their abundance in parallel with parasite development. Interestingly, a number of hereditary haemolytic disorders, known to exert a protective effect on malaria, tend to exacerbate this phenomenon leading to a more precocious and effective opsonization of diseased RBC infected by malaria parasites. The exact definition of band 3 neo-antigens and the mechanism of their surface exposure are still unclear. Also band 3 clusterisation is only superficially understood, new insights about band 3 phosphorylation by Src kinases suggest the presence of a complex regulatory pathway.

Erythrocyte ageing in vivo and in vitro: structural aspects and implications for transfusion.

Erythrocyte transfusion is essential in conditions of large blood loss, of inadequate bone marrow production and of increased erythrocyte breakdown. The structural and biochemical changes that erythrocytes go through during storage, probably associated with the disappearance of up to 30% of the erythrocytes within 24 h after transfusion, are likely to contribute to the transfusion side effects: iron overload, erythrocyte adhesion to the endothelial surface with proinflammatory consequences, autoantibody formation, endothelial damage by released erythrocyte constituents, a hampered microcirculation and oxygen delivery. In vivo, senescent erythrocytes are marked for removal by binding of autologous immunoglobulin G to ageing antigens, which arise by changes in the conformation of the membrane domain of band 3. Also, vesicle formation has been described as an integral part of the erythrocyte ageing process. Comparable changes occur during erythrocyte storage. This review describes the current state of knowledge of the mechanism of erythrocyte ageing in vivo, ageing-related changes occurring during erythrocyte storage in blood bank conditions and their possible relation with the transfusion side effects. In view of the key position of band 3 in the maintenance of erythrocyte structure and function, elucidation of the pathways that control posttranslational modification of band 3 during storage may lead to new approaches towards maintaining ATP concentration and cellular integrity. This review concludes with the challenge to further explore the underlying processes of erythrocyte ageing in order to provide physiologically relevant tools for assessing and predicting erythrocyte homeostasis in vitro and in vivo and thereby to contribute to the development of rational transfusion protocols for various patient categories.