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Excessive binding of the natural anti-α-galactosyl IgG to sickle red cells may contribute to extravascular cell destruction
Abstract
A large proportion of sickle erythrocytes is removed from the circulation by the macrophages of the reticuloendothelial system. In view of the proposed role for natural antibodies in the destruction of normal senescent erythrocytes, we looked for a possible similarity in the antibodies that bind in situ to senescent and sickle cells. Bound IgG molecules were detected by a highly sensitive rosetting antiglobulin test, using K562 myeloid cells. After separation on Stractan density gradients, the 0.6% most dense (senescent) normal cells and the most dense 40% sickle cells displayed membrane-bound IgG as reflected by the high proportion of rosettes formed. No antibody was found on low-density cells of either type. The bound antibodies were readily eluted from both sickle and normal senescent cells by carbohydrates containing alpha-galactosyl residues. These antibodies appear identical to the recently discovered human natural anti-alpha-galactosyl IgG (anti-Gal), an IgG antibody present in high titers in normal sera. Moreover, affinity-purified anti-Gal interacted specifically with sickle and normal cells depleted of the autologous antibodies. A similar pattern of binding to the various erythrocyte subpopulations was observed when the radiolabeled lectin with anti-alpha-galactosyl specificity, Bandeiraea simplicifolia, was used. In vitro phagocytosis of normal and sickle erythrocyte subpopulations correlated with the presence of anti-Gal on these cells. The in situ binding of anti-Gal to a large proportion of sickle erythrocytes may reflect an accelerated physiologic aging process by which immune recognition of prematurely exposed alpha-galactosyl-bearing antigenic sites contributes to shortened cell survival.
... Anti-α-gal antibodies in human sera were first observed in blood pathologies β-thalassemia (10) and sickle cell anemia (11), where distorted red cells were coated in antibodies associated with senescent red cells which contain reduced amounts of sialic acid at their exteriors, thus revealing cryptic α-gal epitopes (12). It was postulated that these antibodies helped remove old, damaged, or misshapen cells via phagocytic macrophages, or destruction via complement. ...
... HKB7 and JEC1 contain aspartic acid and asparagine at V H 50. In HKB7, the projected side chain of V H D50 contributes a hydrogen bond to α-gal (Fig. 3C). Other W33-encoding IGHV germlines contain different residues at position 50, such as arginine (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), glutamate (4-4), or tyrosine (4-28), whose larger side chains might sterically occlude the surface pocket characterized in our structures, and thus impede binding. Indeed, the diversity of α-gal binders observed, combined with insights from other protein-glycan structures (SI Appendix, Tables S7 and S8), suggests that different combinations of residues might achieve low-affinity binding to a variety of carbohydrate ligands. ...
Humans lack the capacity to produce the Galα1–3Galβ1–4GlcNAc (α-gal) glycan, and produce anti-α-gal antibodies upon exposure to the carbohydrate on a diverse set of immunogens, including commensal gut bacteria, malaria parasites, cetuximab, and tick proteins. Here we use X-ray crystallographic analysis of antibodies from α-gal knockout mice and humans in complex with the glycan to reveal a common binding motif, centered on a germline-encoded tryptophan residue at Kabat position 33 (W33) of the complementarity-determining region of the variable heavy chain (CDRH1). Immunoglobulin sequencing of anti-α-gal B cells in healthy humans and tick-induced mammalian meat anaphylaxis patients revealed preferential use of heavy chain germline IGHV3-7, encoding W33, among an otherwise highly polyclonal antibody response. Antigen binding was critically dependent on the presence of the germline-encoded W33 residue for all of the analyzed antibodies; moreover, introduction of the W33 motif into naive IGHV3-23 antibody phage libraries enabled the rapid selection of α-gal binders. Our results outline structural and genetic factors that shape the human anti-α-galactosyl antibody response, and provide a framework for future therapeutics development.
... However, erythrocytes have no nucleus to direct the apoptotic process and the PS display on senescent erythrocytes is the result of the cumulative breakdown of the hemoglobin molecule [1] and is called eryptosis. Normally less than 1% of senescent erythrocytes can be detected in the circulation but in SCD 30-40% of them are senescent [2]. This premature senescence results in many sickle erythrocytes being PS+ and their macrophage elimination being overwhelmed leaving many PS+ erythrocytes in the circulation to eventually be removed by inflammatory necrosis. ...
... As previously noted 30-40% of sickle erythrocytes are prematurely senescent [2] and unless they are rapidly removed these senescent erythrocytes adhere to the endothelium via a variety of receptors. The significance of this is supported by an in vitro study showing that 36% of the endothelial adhesion of sickle erythrocytes can be prevented by using annexin V to cloak the PS molecules [3] and prevent access to its endothelial receptor thrombospondin (TBS) [10]. ...
A sickle cell crisis is a painful and dangerous condition that defies effective treatment but fortunately it usually terminates spontaneously and patients spend far more time crisis free than in its painful throes. This suggests that an unstable physiologic balance exists between steady state sickle cell disease (SCD) and the crisis state and if this is so a therapeutic nudge during a crisis may help to terminate it. Annexin V may be able to provide this push. The phosphatidylserine (PS) molecules normally appear on the surface of senescent erythrocytes where they are recognized by macrophages and rapidly removed so that normally only about 1% are present in the circulation but in SCD 30-40% are prematurely senescent and their removal is delayed. The PS+ sickle erythrocytes remaining in the circulation adhere to the endothelium and their exposed PS acts as a platform for the initiation of the coagulation cascade that is responsible for clot propagation. Annexin V's great affinity for PS allows it to bond to it forming a shield that blocks both of these actions suggesting that its therapeutic administration during a sickle crisis may be able to hasten its termination.
Copyright © 2015. Published by Elsevier Ltd.
... In sickle cell anemia (SCA) the sickle erythrocytes experience premature senescence due to their mutant hemoglobin molecules and this causes band 3 clusters to form on their surface that are recognized by natural band 3 antibodies [4]. The significance of this regarding both anemia and endothelial adhesion in SCA is clear when you consider that in normal blood only 1% or less of erythrocytes have bound antibody compared to 30-40% in those with SCA [5]. The premature senescence of sickle erythrocytes also causes the apoptosis molecule, phosphatidylserine, to appear on the erythrocytes surface and it also adheres to the vascular endothelium [6]. ...
... This suggests that there may be some type of biologic feedback control that limits their production and that in steady state SCA the cluster level exceeds this limit. The previously noted 30 fold increase in the presence of antibodies on sickle erythrocytes [5] could reasonably explain why ...
The band 3 molecule is an anion channel for bicarbonate in the erythrocyte’s membrane that assumes a different role in senescent erythrocytes where it adopts a cluster configuration. These band 3 clusters expose previously hidden antigenic adhesive peptides which are recognized by natural band 3 antibodies that label their erythrocytes for reticuloendothelial elimination and prevent their endothelial adhesion. The sickle erythrocyte’s abnormal hemoglobin molecules cause them to experience premature senescence resulting in increased numbers of clusters, a portion of which remain uncloaked by antibodies and adhere to the vascular endothelium. The uncloaked clusters result from a deficiency of band 3 antibodies that are present in sickle cell anemia. The hypothesis presented is that the band 3 antibodies are insufficient in number to cloak all of the adhesive peptides present on cluster+ sickle erythrocytes and this is responsible for a portion of their adhesive pathology
... It is of interest to note that the initial discovery of anti-Gal was on red blood cells (RBC) of patients with b-thallassaemia, 30 on human normal senescent RBC 1,31 and on sickle cell anaemia RBC. 32 It is possible that a cryptic antigen capable of binding anti-Gal is exposed on human RBC that are~120 days old, or on thalassaemia and sickle cell anaemia RBC where this antigen is exposed at a younger age of the RBC. 1,[30][31][32] The amount of this cryptic antigen on RBC is very low, resulting in the binding of only a few hundred anti-Gal IgG molecules and its molecular structure has yet to be characterized. ...
... 32 It is possible that a cryptic antigen capable of binding anti-Gal is exposed on human RBC that are~120 days old, or on thalassaemia and sickle cell anaemia RBC where this antigen is exposed at a younger age of the RBC. 1,[30][31][32] The amount of this cryptic antigen on RBC is very low, resulting in the binding of only a few hundred anti-Gal IgG molecules and its molecular structure has yet to be characterized. ...
Anti-Gal is the most abundant natural antibody in humans constituting ~1% of immunoglobulins. Anti-Gal is naturally produced also in apes and Old World monkeys. The ligand of anti-Gal is a carbohydrate antigen called the "α-gal epitope" with the structure Galα1-3Galβ1-4GlcNAc-R. The α-gal epitope is present as a major carbohydrate antigen in nonprimate mammals, prosimians and New World monkeys. Anti-Gal can contribute to several immunological pathogeneses. Anti-Gal IgE produced in some individuals causes allergies to meat and to the therapeutic monoclonal antibody cetuximab, all presenting α-gal epitopes. Aberrant expression of the α-gal epitope or of antigens mimicking it in humans may result in autoimmune processes, as in Graves' Disease. α-Gal epitopes produced by Trypanosoma cruzi interact with anti-Gal and induce "autoimmune like" inflammatory reactions in Chagas Disease. Anti-Gal IgM and IgG further mediate rejection of xenografts expressing α-gal epitopes. Because of its abundance, anti-Gal may be exploited for various clinical uses. It increases immunogenicity of microbial vaccines (e.g., flu vaccine) presenting α-gal epitopes by targeting them for effective uptake by APC. Tumor lesions are converted into vaccines against autologous tumor associated antigens by intratumoral injection of α-gal glycolipids which insert into tumor cell membranes. Anti-Gal binding to α-gal epitopes on tumor cells targets them for uptake by APC. Accelerated wound healing is achieved by application of α-gal nanoparticles which bind anti-Gal, activate complement, recruit and activate macrophages that induce tissue regeneration. This therapy may be of further significance in regeneration of internally injured tissues such as ischemic myocardium and injured nerves. This article is protected by copyright. All rights reserved.
... Moreover, sickled RBCs under hypoxia show expedited engulfment by macrophages compared to the case of non-sickled RBCs either under normoxic or hypoxic conditions. These phenomena are possibly associated with multiple biochemical mechanisms, including hypoxia-enhanced PS exposure (53,54), Band 3 clustering (26,(55)(56)(57)(58)(59), and CD47 expression (25, 60-62) on SS RBC membrane, thereby facilitating the recognition and engulfment of sickle cells by macrophages (59,63,64). In addition to the interfacial biochemical factors, our study shows that sickled SS RBCs exhibit altered biophysical properties (abnormal shapes and increased rigidity) as opposed to AA RBCs and non-sickled SS RBCs, which may also play the analogous role in promoting the elimination of RBCs by the macrophages (28,(65)(66)(67). ...
The spleen clears altered red blood cells (RBCs) from circulation, contributing to the balance between RBC formation (erythropoiesis) and removal. The splenic RBC retention and elimination occur predominantly in open circulation where RBCs flow through macrophages and inter-endothelial slits (IESs). The mechanisms underlying and interconnecting these processes significantly impact clinical outcomes. In sickle cell disease (SCD), blockage of intrasplenic sickled RBCs is observed in infants splenectomized due to acute splenic sequestration crisis (ASSC). This life-threatening RBC pooling and organ swelling event is plausibly triggered or enhanced by intra-tissular hypoxia. We present an oxygen-mediated spleen-on-a-chip platform for in vitro investigations of the homeostatic balance in the spleen. To demonstrate and validate the benefits of this general microfluidic platform, we focus on SCD and study the effects of hypoxia on splenic RBC retention and elimination. We observe that RBC retention by IESs and RBC-macrophage adhesion are faster in blood samples from SCD patients than those from healthy subjects. This difference is markedly exacerbated under hypoxia. Moreover, the sickled RBCs under hypoxia show distinctly different phagocytosis processes from those non-sickled RBCs under hypoxia or normoxia. We find that reoxygenation significantly alleviates RBC retention at IESs, and leads to rapid unsickling and fragmentation of the ingested sickled RBCs inside macrophages. These results provide unique mechanistic insights into how the spleen maintains its homeostatic balance between splenic RBC retention and elimination, and shed light on how disruptions in this balance could lead to anemia, splenomegaly, and ASSC in SCD and possible clinical manifestations in other hematologic diseases.
... Haemolysis ensuing to the damaged red cell membrane could be intravascular or extravascular. The former event occurs as a result of the lysis of complement sensitive red cells (5) and haemoglobin lost during sickling-induced damage to the membrane of the RBCs (6,7) The latter event on the other hand mentioned, happens by phagocytosis of red cells that have gone through sickling (8,9) and physical entrapment of rheologically compromised red blood cells (10) . Increased vulnerability to mechanically induced cell breakdown has been reported in-vitro and in sickle cell patients undergoing strenuous physical activity (7) . ...
The study was conducted to examine the haematological profile in patients with
sickle cell disorder, to study the Mentzer Index and Srivastava Index in patients with sickle cell disorder and to assess the prevalence of iron deficiency anaemia with sickle cell
disorders.
... However individuals who express these variants are phenotype with RhD positive, but develop anti-D, lead to decreased survival of erythrocytes, because such individuals become dependent blood transfusion and higher risk alloimmunization. These results indicate the serological typing of red blood cells cannot distinguish between variant gene products RhD, resulting in a later alloimmunization and a greater commitment survival of erythrocytes, once these patients have already changed the erythroid series sickle shaped and is also associated with changes in Rh protein [8,9]and are expressed in them a greater amount of adhesion molecules and immunoglobulins [10,11], It may be associated with an increased susceptibility for transfusion in these individuals. ...
... A subpopulation of erythrocytes in SCD is bound to immunoglobulin G (IgG) (20,(69)(70)(71). Cyclic deoxygenation or oxidant stress in sickle cell disease indicated by malondialdehyde production stimulates binding of IgG to the red cell surface (20,72). ...
Overview of Hemolysis in Sickle Cell Disease and Thal Assemia: Anemia is the most basic clinical characteristic of sickle cell disease and thalassemia. In sickle cell disease, the polymerization of sickle hemoglobin (HbS) causes profound changes in the integrity and viability of the erythrocyte, leading to both extravascular and intravascular hemolysis. The lifespan of the erythrocyte in sickle cell disease is often shortened to less than one-tenth of normal. In β-thalassemia intermedia and major, but not in sickle cell disease, a substantial portion of the hemolysis occurs in the intramedullary space before the developing erythrocytes can even exit the bone marrow, referred to as ineffective erythropoiesis. In either case, erythropoiesis is markedly increased, but insufficient to compensate completely for the accelerated hemolysis, resulting in chronic anemia. This chapter examines the mechanisms that give rise to the accelerated hemolysis characteristic of these hemoglobinopathies and considers emerging data suggesting that chronic intravascular hemolysis produces endothelial dysfunction and a progressive vasculopathy. The latter mechanism of disease contributes to a clinical subphenotype of complications shared by many of the hemolytic anemias, including pulmonary arterial hypertension, cutaneous leg ulceration, priapism, and perhaps stroke. The mechanisms and consequences of hemolysis differ by two main anatomical compartments: extravascular hemolysis, which primarily involves phagocytosis by macrophages in the reticuloendothelial system, and intravascular hemolysis, which occurs within the blood vessel lumen. Approximately two-thirds of hemolysis in sickle cell disease is extravascular and one-third intravascular.
... The complete absence of anti-Gal clones capable of reacting with self blood group B antigen suggests that the immune tolerance mechanism exerts a powerful effect, preventing the generation of antibodies to self antigens. Related to this study are our recent observations on the contribution of anti-Gal to the immune-mediated destruction of senescent and pathologic human red cells (10,(32)(33)(34) . These studies have suggested that Gala l--*3Gal epitopes are present on human red cells in small amounts, and in a cryptic form. ...
A well-defined antigen/antibody system was used to evaluate the effect of immune tolerance on the spectrum of specificities of natural antibodies. The antibody used in this study, anti-Gal, is a naturally occurring, polyclonal IgG that constitutes 1% of the circulating IgG in humans. We have previously shown that anti-Gal, purified from AB sera, specifically interacts with glycosphingolipids bearing a Gal alpha 1----3Gal epitope, but not with the closely related B antigen in which the penultimate galactose of the Gal alpha 1----3Gal epitope is fucosylated Gal alpha 1----3(Fuc alpha 1----2)Gal. This narrow specificity was assumed to be the result of an effective immune tolerance mechanism that prevents the expression of antibody clones that can recognize both the Gal alpha 1----3Gal and the self B epitopes. If the assumption that immune tolerance determines the range of anti-Gal specificity is correct, then anti-Gal from individuals lacking the B antigen (A and O blood types) would be expected to interact with both Gal alpha 1----3Gal and Gal alpha 1----3(Fuc alpha 1----2)Gal epitopes. In this study, anti-Gal from the serum of individuals of various blood types was purified by affinity chromatography on Gal alpha 1----3Gal adsorbent and tested for its reaction with the B antigen. Whereas anti-Gal from AB and B individuals only reacted with Gal alpha 1----3Gal epitopes, anti-Gal from A and O individuals reacted with both Gal alpha 1----3Gal and B epitopes. Furthermore, it was determined that the majority of anti-B reactivity in A and O individuals is in fact anti-Gal antibodies capable of recognizing both Gal alpha 1----3Gal and B epitopes. It can be concluded from these results that immune tolerance accurately controls the spectrum of natural antibody specificities by preventing the production of antibody clones that can interact with self antigens.
... On the other hand, large amounts of naturally occurring antibodies specifically against ␣-gal epitope are present in the serum of humans who are not immune compromised (13,17). The binding of antibody to ␣-gal epitope activates the complement system and leads to the hyperacute rejection (HAR) of the ␣-gal-expressing cells and tissues (3,10,12,15,16,32,49). ...
Engineering cancer cells to express heterologous antigen α-gal and induce the destruction of tumor cells depending on the complement cascade may be a promising strategy of tumor therapy. However, the feasibility and effect of using α-gal to induce colorectal adenocarcinoma cell lines cytolysis is not yet known. In this study, we evaluated α-gal expression's ability to sensitize human colorectal adenocarcinoma cell lines to complement attack in cell lines LoVo, SW620 and Ls-174T. Nearly all α-gal -expressing LoVo and SW620 cells were killed by human normal serum (NHS), but α-gal expression Ls-174T cells showed no significant lysis. We analyzed the expression levels of membrane-bound complement regulatory proteins (mCRPs) on the three cell lines, and their protective role in α-gal-mediated activation of the complement. LoVo show no expression of any of the three proteins. CD59 was strongly expressed by SW620 and Ls-174T. CD46 and CD55 varied between the two cell lines, CD46 on SW620 was only half the intensity of CD46 on Ls-174T. Ls-174T showed a notable expression of CD55, while expression of CD55 on SW620 was not detected. The sensitivity of Ls-174T expressing α-gal to NHS greatly increased following the down-regulation of CD46 and CD55 with shRNA. However, there is no increase in cell killing when CD59 expression was diminished. Our findings suggest that the use of α-gal as antigen to induce tumor cell killing may be a potential therapeutic strategy in colon cancer, and that CD55 plays a primary role in conferring resistance to lysis.
... Extravascular hemolysis occurs by phagocytosis of red cells that have undergone sickling and physical entrapment of rheologically compromised red cells (4)(5)(6). Increased susceptibility to mechanically induced cell fragmentation has been documented in vitro and in sickle cell patients undergoing vigorous exercise (3). ...
Background:
Sickle cell disease is a genetic disorder of hemoglobin causing myriad of pathology including anemia. The purpose of this study was to evaluate the baseline values of steady state hemoglobin and packed cell volume as a guide to managing the early recognition of hemolytic crises in sickle cell anemia.
Methods:
A cross-sectional study was conducted among the sickle cell patients attending the Sickle Cell clinic of Lagos State University Teaching Hospital, Ikeja. A blood sample of 4.5 ml blood was collected from each participant for hemoglobin concentration and packed cell volume. All blood samples were also screened for HIV and hemoglobin phenotypes were done using cellulose acetate hemoglobin electrophoresis at pH 8.6.
Results:
A total of 98 subjects in steady state were recruited, consisting of 53 (54.1%) females and 45 (45.9%) males. The overall means were 7.92±1.49 and 24.46±4.76; a female mean of 7.73±1.45; 23.89±4.60, and a male mean of 8.14±1.54 and 25.14±4.91 were obtained for hemoglobin and packed cell volume, respectively. Sixty - nine of the 98 (70.40%) subjects have been previously transfused with blood.
Conclusion:
The mean hemoglobin concentration and packed cell volume in males was higher than females. The overall mean was lower than what was expected for age and sex. Over two-third of sickle cell anemia population had been transfused.
... In the process of human red blood cell ageing, cryptic α-gal epitopes are exposed on the cell surface by the action of macrophage hydrolases [28,51]. For this reason, a few hundred α-gal epitopes are present on the surface of normal senescent and sickle red blood cells (e.g., thalassemic cells) [28,52]. Naturally occurring anti-Gal antibodies bind this epitope, enhancing the phagocytosis of red blood cells by reticuloendothelial macrophages and thus, their removal from circulation [28,53]. ...
The glycan chains attached to cell surfaces or to single proteins are highly dynamic structures with various functions. The glycan chains of mammals and of some microorganisms often terminate in sialic acids or α-1,3-galactose. Although these two sugars are completely distinct, there are several similarities in their biological and medical importance. First, one type of sialic acid, N-glycolylneuraminic acid, and the galactose bound by an α-1,3-linkage to LacNAc, that forms an α-gal epitope, were both eliminated in human evolution, resulting in the production of antibodies to these sugars. Both of these evolutionary events have consequences connected with the consumption of foods of mammalian origin, causing medical complications of varying severity. In terms of ageing, sialic acids prevent the clearance of glycoproteins and circulating blood cells, whereas cryptic α-gal epitopes on senescent red blood cells contribute to their removal from circulation. The efficiency of therapeutic proteins can be increased by sialylation. Another common feature is the connection with microorganisms since sialic acids and α-gal epitopes serve as receptors on host cells and can also be expressed on the surfaces of some microorganisms. Whereas, the sialylation of IgG antibodies may help to treat inflammation, the expression of the α-gal epitope on microbial antigens increases the immunogenicity of the corresponding vaccines. Finally, sialic acids and the α-gal epitope have applications in cancer immunotherapy. N-glycolylneuraminic acid is a powerful target for cancer immunotherapy, and the α-gal epitope increases the efficiency of cancer vaccines. The final section of this article contains a brief overview of the methods for oligosaccharide chain synthesis and the characteristics of sialyltransferases and α-1,3-galactosyltransferase.
... The former results from the lysis of complement-sensitive red cells [1] and haemoglobin lost during sickling-induced membrane damaged. [2,3] The latter, occurs by phagocytosis of red cells that have undergone sickling [4,5] and physical entrapment of rheologically compromised red cells.[6] Increased susceptibility to mechanically induced cell fragmentation has been documented in-vitro and in sickle cell patients undergoing vigorous exercise [3]. ...
Sickle cell disease is a genetic abnormality involving the haemoglobin. Although, it is primarily a red cell disorders, the white blood cells and platelets are also affected by the mutation. The consequent haemoglobin S causes polymerization of haemoglobin resulting in haemolysis and anaemia. This study aims to provide baseline haematological values in sickle cell disease patients in steady state and compare the deviation from haemoglobin phenotype AA control values.
A case-control study was conducted amongst homozygous sickle cell patients attending the sickle cell clinics of Lagos State University Teaching Hospital Ikeja and haemoglobin phenotype AA controls. About 4.5mls of blood sample was collected from each participant for full blood count analysis. All blood samples were screened for HIV and haemoglobin phenotypes confirmed using cellulose acetate haemoglobin electrophoresis at pH 8.6.
A total of 103 cases and 98 controls were enrolled. The overall mean haemoglobin concentration for cases was 7.93 ± 1.47 g/dl, packed cell volume 24.44 ± 4.68%, mean cell volume 81.52 ± 7.89 fl, and mean cell haemoglobin 26.50 ± 3.20 pg. While for controls, mean haemoglobin concentration was 13.83 ± 1.32 g/dl, packed cell volume 43.07 ± 3.95%, mean cell volume 86.90 ± 4.69 fl, and mean cell haemoglobin 28.50 ± 1.34 pg. The overall mean white blood cell counts for the cases was 10.27 ± 3.94 *103/μl and platelet counts of 412.71 ± 145.09*103/μl. While white blood cell count for the controls was 5.67 ± 1.59*103/μl and platelet counts of 222.82 ± 57.62*103/μl.
Homozygous sickle cell disease patients have lower values of red cell parameters, but higher values of white cell and platelets counts compared to haemoglobin phenotype AA controls.
... While a great deal is known about erythropoiesis and the role played by a variety of cytokines and growth factors in this process (Barosi 1994;Miller et al 1994;Jacobsen 1995;Roeder et al 1998), the mechanisms involved in destruction of erythrocytes are not well understood. It is believed that aging erythrocytes are trapped in the spleen and bone marrow where phagocytic cells of the reticuloendothelial system remove them (Galili et al 1986;Connor et al 1994). How aging erythrocytes are recognized by the phagocytes is not clear, though there are several speculations in the literature about the process of senescence of erythrocytes and the changes that may lead to their recognition by the reticuloendothelial system (Hensley et al 1989;Kay et al 1989;Kosower 1993). ...
... This class of antibodies, which are present in the serum without a known antigenic stimulation [24][25][26], are a component of the normal humoral arm of the immune system in human. It has been proposed that natural antibodies participate in enhancement of: (1) opsonisation of foreign antigens [27]; (2) independent B-cells and T-cells respond to foreign antigens [28,29]; (3) clearance of catabolic products and soluble immune complexes [30][31][32][33][34]; and (4) increased protection against infection [35][36][37]. Autoantibodies are a subgroup of natural antibodies that react with self-antigens [24][25][26]38]. ...
Low maternal plasma protein Z (PZ) concentrations were reported in patients with pre-eclampsia (PE), a small for gestational age (SGA) neonate, and a fetal demise (FD). Anti-protein Z antibodies (APZ-AB) have been proposed as a possible underlying mechanism leading to low plasma PZ concentrations. The objective of this study was to determine the maternal plasma concentration of APZ-AB in women with a normal pregnancy, and patients with PE, an SGA neonate or a FD.
A cross-sectional study included women in the following groups: (1) non-pregnant women (n = 45); and pregnant women with: (2) normal pregnancies (n = 70); (3) PE (n = 123); (4) SGA neonates (n = 51); and (5) a FD (n = 51). Plasma concentrations of anti-protein Z IgM and IgG antibodies were measured by ELISA. Elevated APZ-AB was defined as >75th, 90th and 95th percentile of the normal pregnancy group. Non-parametric statistics were used for analyses.
(1) Patients with an SGA neonate had a higher median maternal plasma IgG APZ-AB concentration than women with normal pregnancies (p < 0.001), and patients with PE (p < 0.001) or with a FD (p = 0.001). (2) The proportion of patients with a maternal plasma IgM APZ-AB concentration >90th percentile was higher in the SGA group than in the PE group (p = 0.01). (3) Patients with PE maternal plasma IgM APZ-AB concentration >90th percentile had a higher rate of villous thrombosis (p = 0.03) and persistent muscularization of basal plate arteries (p = 0.01) than those with IgM APZ-AB concentration <90th percentile; and (5) Patients with FD and maternal plasma IgM APZ-AB concentration >90th percentile had a higher rate of umbilical phlebitis and arteritis than those with IgM APZ-AB concentration <90th percentile (p = 0.003).
(1) Patients with SGA neonates have a higher median plasma concentration of IgG APZ-AB than normal pregnant women, or patients with PE or FD; and (2) maternal plasma IgM APZ-AB concentration >90th percentile was associated with vascular placental lesions in patients with PE, but not in those with an SGA neonate, suggesting that in a subset of patients, these antibodies can be associated with abnormal placentation and pregnancy complications.
... Another question still unanswered is the role of anti-Gal antibodies. They have been considered involved in removal of altered erythrocytes (Galili et al., 1984Galili et al., , 1985 Galili, Clark & Shohet, 1986) and in the recognition of tumour cells (Towbin et al., 1987; Castronovo et al., 1989) since evidence for the presence of oligosaccharides terminating with Galal-3Gal has been found in senescent erythrocytes and in cancer cells. Their implication in the pathogenesis of connective tissue diseases and in autoimmunity in general is less defined. ...
Employing radioimmunoinhibition assays with distinct oligosaccharides as inhibitors, this study demonstrates that the epitope recognized on mouse laminin by sera from patients with systemic sclerosis (scleroderma) is a terminal galactosyl (alpha 1-3)-galactose disaccharide. The reaction with this alpha-digalactose was further confirmed when the sera were tested in radioimmunoassay (RIA) binding assay and in ELISA with synthetic galactose alpha 1-3 galactose coupled to human serum albumin. The circulating antibody appeared restricted to the IgG class and mostly to the subclass IgG3 and IgG4. Antibodies with the same specificity can be found in patients with American cutaneous leishmaniasis and Chagas disease; however, whilst in these diseases the antibody production is triggered by antigenic determinants present on the surface of the parasites, the events eliciting their appearance in systemic sclerosis are unknown.
This book is a completely revised new edition of the definitive reference on disorders of hemoglobin. Authored by world-renowned experts, the book focuses on basic science aspects and clinical features of hemoglobinopathies, covering diagnosis, treatment, and future applications of current research. While the second edition continues to address the important molecular, cellular, and genetic components, coverage of clinical issues has been significantly expanded, and there is more practical emphasis on diagnosis and management throughout. The book opens with a review of the scientific underpinnings. Pathophysiology of common hemoglobin disorders is discussed next in an entirely new section devoted to vascular biology, the erythrocyte membrane, nitric oxide biology, and hemolysis. Four sections deal with α and β thalassemia, sickle cell disease, and related conditions, followed by special topics. The second edition concludes with current and developing approaches to treatment, incorporating new agents for iron chelation, methods to induce fetal hemoglobin production, novel treatment approaches, stem cell transplantation, and progress in gene therapy.
A subset of sickle cells have an increased density at the reticulocyte stage of development, indicating that they are either abnormally dense upon release from the bone marrow or become dense quickly in the circulation. These cells are of interest because they most likely have severely disrupted cation regulation and a short lifespan. Based on the distribution of fetal hemoglobin (HbF) in the density fractions of sickle red blood cells (RBCs) and in vitro studies of cellular K+ loss, it seems likely that HbF content is an important in vivo determinant of dense cell formation. In this study, we tested the hypothesis that young, dense cells have low HbF content. Sickle RBCs were first separated into light and dense fractions. Reticulocytes were isolated from unfractionated cells and from each density fraction with an immunomagnetic technique directed against transferrin receptors (TfR) and assayed for the percentage of HbF and K+/Hb ratio. TfR+ reticulocytes isolated from unfractionated cells had a much lower HbF content when compared with all the unfractionated RBCs. This is most likely caused by enrichment of F cells because of a longer circulation life span. Heavy TfR+ reticulocytes had a K+/Hb ratio similar to that measured in the entire dense population and contained very low levels of HbF, averaging 2.5% of the level in all RBCs, 11.7% of the level in all TfR+ reticulocytes, and 4.0% of the level in all dense RBCs. These findings suggest that TfR+ dense cells derive predominantly from non-F cells. Furthermore, the amount of HbF in the circulating dense cells suggests that many of these cells do not derive from the TfR+ dense cells.
We have previously reported that sickle erythrocytes sedimenting at high specific density after gradient centrifugation exhibit increased IgG binding in vivo as compared with low-density paired samples. We have performed the present study to determine whether the opsonization of dense sickle cells in vivo could also involve autologous IgM, IgA, and complement. IgA, IgM, and complement binding in vivo to the surface of density-separated sickle erythrocytes was detected by flow cytometric analyses. IgM and complement C3 fragment binding was detected primarily on high-density sickle erythrocytes. With the exception outlined below, IgA binding was detected for all sickle cell fractions that sediment at densities > 1.085 g/mL. IgM, IgA, and complement C3 fragment binding was increased on high-density sickle erythrocytes as compared with low-density paired samples and exceeded that binding to normal erythrocytes by 30% +/- 10% (mean +/- range), 50% +/- 10%, and 41% +/- 5%, respectively. Two-color flow cytometry indicates that high-density sickle cell fractions contain at least two heterogeneous RBC subsets. One is an RBC subset that binds IgA in combination with IgM and C3, and the second subset is devoid of IgA yet binds IgM and C3. These findings indicate that high-density sickle cells exhibit a greater heterogeneity than has been reported in previous studies, which is based on autologous Ig binding in vivo; and suggest that RBC components of this most severely dehydrated sickle cell subpopulation could have heterogeneous origin and pathophysiologic significance. Although the functional role of IgA binding to human RBCs is unclear, our findings that IgM and complement bind to the same high- density sickle cell fractions suggest that both the IgM and the sickle erythrocyte-bound IgG determined in previous studies could mediate the deposition of complement on dense sickle cells in vivo. These findings support the hypotheses that irreversibly sickled cell-enriched high- density sickle RBC subpopulations could be removed from the circulation by erythrocyte phagocytosis that is enhanced by the presence of complement.
Gal-α-1,3-Gal (α-Gal) epitope is a carbohydrate structure, which is expressed on cell membranes of almost all the mammals except some species of monkeys and human, as well as causes a hyperacute immune response during xenotransplantation. This review briefly describes the issues of the evolution of α-Gal epitope, its distribution in the kingdom of animals, biological role and problems of using prostheses of porcine origin in medical practice. For many reasons, this species of animals is the most suitable to obtain the bioprostheses of cardiac valves, vessels and bioscaffolds, however, due to the high α-Gal epitope expression in their body a special treatment to eliminate it is required. This review attempts to summarize the results on studying the α-Gal epitope expression after low-temperature treatment, since it is widely used to store bioprostheses prior to implantation.
Some organs of the body are uniquely associated with specific diseases resulting from disturbance or failure of their own structure or function. For example, renal failure is the consequence of disorders of the kidneys, and myocardial infarction results from structural damage to the heart. But the spleen is in many respects unique, and the disorders that are most closely associated with the spleen have their etiological origins elsewhere. For example, hereditary disorders of the red blood cell membrane lead to red blood cell destruction by the spleen. Likewise, in autoantibody-mediated blood cell damage, the spleen may play both a causal and a consequential role. In these conditions, the spleen produces the autoantibodies that initiate cell damage, and also provides the filtration mechanism that destroys the antibody-sensitized cells.
One convenient classification of hemoglobin disorders dichotomizes these conditions into mutations that either change the primary structure of globin, called hemoglobinopathies, or reduce the expression of globin genes, called thalassemia. Rarely, a single mutation affects both the structure and expression level of the affected gene, and these have been called thalassemic hemoglobinopathies. Although at the level of the abnormal erythrocyte, many hemoglobinopathies and thalassemias can have similar pathophysiological effects, and result in similar clinical features, such generalizations do not apply to all hemoglobin disorders. For example, only sickle hemoglobin (HbS) polymerizes, triggering many features of sickle cell disease, and extreme ineffective erythropoiesis is characteristic of β thalassemia major. The next four chapters consider how hemoglobin disorders can perturb vascular biology, the erythrocyte membrane, nitric oxide biology, and the lifespan of the affected red cell. A final chapter brings up to date the animal models that permit certain detailed study of this pathobiology. All these chapters are new to this edition. In sickle cell disease and thalassemia, chronic hypoxia and hemolysis require vascular tone and flow adaptations. In sickle cell disease, red cell deformability is sensitive to intravascular oxygen tension as deoxygenation results in HbS polymerization, erythrocyte sickling, and vasoocclusion. Vasoocclusive events in sickle cell disease result in reperfusion injury characterized by excessive oxidant generation, endothelial activation and dysfunction, and inflammation. Similarly, in thalassemia, red cell abnormalities caused by precipitation of excess globin chains and oxidative stress lead to hemolysis and reduced red cell deformability and contribute to vascular pathobiology.
The carbohydrate structure Galαl-3Galß1-4G1cNAc-R (termed the α-galactosyl epitope) and the natural antibody which interacts specifically with this epitope (termed anti-Gal) display a unique pattern of distribution in primates. The α-galactosyl epitope is abundantly expressed on red cells and nucleated cells of prosimians and New World monkeys as well as on cells of nonprimate mammals. It is absent, however, from Old World monkeys, apes, and humans. In contrast, anti-Gal is produced in large amounts in Old World monkeys, apes, and humans but is absent from New World monkeys, prosimians, and nonprimate mammals. The studies reported on in this chapter: (1) identify the α-galactosyl epitope as the “B-like” antigen reported by Landsteiner 70 years ago to be present on New World monkey red cells; (2) describe the reciprocity in the distribution of the α-galactosyl epitope and of anti-Gal in primates; (3) discuss the molecular aspects of the evolutionary event that led to suppression of α-galactosyl epitope expression and the appearance of anti-Gal in ancestral Old World primates (i.e., Old World monkeys and apes); and (4) discuss the pathophysiologic outcomes of the interaction between the α-galactosyl epitope and anti-Gal in the areas of red cell aging, xenotransplantation and autoimmunity. Finally, this chapter describes the potential exploitation of anti-Gal and the α-galactosyl epitope for the augmentation of viral and tumor vaccine immunogenicity.
Maintenance of tissue function includes disposal of nonfunctional, senescent, and aberrant cells. Removal of these cells is a tissue homeostatic process that occurs in one way or another in any multicellular organism with somatic and germline tissues. Vertebrates with a constant body weight and tissues of mesenchymal and mesodermal origin had to acquire tools to eliminate cells by means other than mummification or release to the surroundings. Our knowledge of specific clearance mechanisms is very limited. Our ignorance is in part due to the reliability with which the clearance systems work. There is to my knowledge no known disease or metabolic error that can be traced to an improper clearance system. In contrast to this, a large number of diseases are initiated by an overreaction of a particular clearance mechanism. Consider, for example, the various autoimmune diseases where excessive clearance is mediated either by cellular autoimmunity or by autoantibodies, phagocytes, and in part by complement. Thus, the humoral and cellular defense systems are the primary and definitely the most versatile tools for a selective clearance also of senescent and altered self. The recent insights into the generation of diversity among antibodies (Tonegawa, 1985; Alt et al., 1987), the immunological network (Jerne, 1984; Bottomly, 1984; Hooper, 1987), the persistence of autoreactive B cells in ontogeny (Bona, 1988; Benner et al., 1982), the existence of naturally occurring autoantibodies in healthy subjects (Lutz and Wipf, 1982; Guilbert et al., 1982; Lutz et al., 1984), and the understanding of complement regulation (Müller-Eberhard and Schreiber, 1980; Fearon and Wong, 1983; Schifferli et al., 1986) have paved a road into the complexity of tissue homeostasis and clearance.
A normal immune system contains autoantibodies against self-antigens, termed as termed natural autoantibodies (NAAbs). NAAbs are found in many species, and appear to be conserved through evolution. The physiological significance of NAAbs is not well understood; however, it is postulated that NAAbs have various biological roles, such as first line protection against infection, immunoregulation, and clearance of altered self antigens. Recently, we have detected NAAbs against insulin in healthy cats while developing an enzyme-linked immunosorbent assay (ELISA) for determining feline serum insulin concentration. Our study showed that feline NAAbs against insulin were polyclonal/polyreactive and had a high affinity and binding capacity. In this review, we outline the nature of NAAbs and the methods for detecting and analyzing of NAAbs, including ELISA for screening, affinity chromatography, competitive radioimmunoassay, and epitope analysis.
This chapter discusses the methods of detection of alpha-galactosyl (anti-Gal) autoantibodies. Anti-Gal comprise a large proportion of the several hundred immunoglobulin G molecules that bind in vivo to normal human senescent red cells, to red cells in β-thalassemia and in sickle cell anemia patients and mediate the phagocytosis of these cells by macrophages. The unique pattern of distribution of anti-Gal and the α-galactosyl epitope in mammals and the studies of the biosynthesis of the α-galactosyl lead to the understanding of the major contribution of anti-Gal to the rejection of xenografts in humans and primates. Anti-Gal are natural antibodies unique to humans and Old World primates. These antibodies appeared in ancestral primates subsequent to the evolutionary suppression of α-galactosyl epitope expression. Anti-Gal contribute to the removal of senescent red cells from the circulation, stimulate thyrocytes in patients with Graves' disease by interaction with ligands aberrantly expressed on these cells, and prevent the transplantation of xenografts from nonprimate mammals into humans.
Sera from patients with American cutaneous leishmaniasis and Chagas disease and from monkeys infected with either Trypanosoma cruzi or Trypanosoma rhodesiense show, in RIAs, strong binding to mouse laminin. A distinct although weaker binding activity is also detected in normal human sera. The antibodies recognize a common carbohydrate epitope present on mouse laminin, which was assigned to a terminal galactosyl(alpha 1-3)-galactose group. Distinct crossreactions were observed with some other basement membrane proteins, rabbit glycosphingolipids, defucosylated human B blood group substance and components produced by some human tumor cells. Only little activity was, however, found on laminin obtained from human placenta. The data indicate that the antibodies arising in infectious diseases are stimulated by similar carbohydrate epitopes present on the surface of parasites. Tissue-specific occurrence of such epitopes may exist and explain the involvement of distinct tissues in autoimmune disorders.
In this paper a novel highly birefringent neodymium-doped microstructured optical fiber is presented. The modal properties of the structure are analyzed numerically using a fully vectorial finite difference method. Results obtained are compared with experimental near-field measurements. The emission properties of the structure have also been investigated. The emission cross-sections at 900, 1064 and 1334 nm have been measured. Laser action has been achieved at a wavelength of 1064 nm in the simplest configuration, based on Fresnel light reflection at both ends of the fiber. The optical conversion efficiency was 9% and the slope efficiency reached 16%. A laser configuration with an external fiber Bragg grating has also been studied; promising results obtained for this configuration suggest that such a laser can find applications in sensing.
Anti-insulin immunoglobulin G (IgG) has been found in the sera of healthy cats. To determine the concentrations of these antibodies, an enzyme-linked immunosorbent assay (ELISA) for anti-insulin IgG was developed. ELISA maintained the linearity of a standard concentration line between 67.5 and 2160ng/ml. The coefficients of variances (CVs) of intra-assays in two different plasma samples were 4.0% and 3.7%, respectively. The inter-assay CVs in two different plasma samples were 5.1% and 6.9%, respectively. The dilution curves of two samples were rectilinear. Anti-insulin IgG was detected in all 84 of the healthy cats that were tested. Plasma anti-insulin IgG concentrations ranged from 80 to 1578μg/ml, with a median concentration of 221μg/ml, and this value correlated positively with total plasma IgG concentrations (r=0.383, p<0.01). In an intravenous glucose tolerance test, plasma anti-insulin IgG concentrations did not alter, even with changes in plasma glucose and insulin concentrations. The ELISA that was developed was able to determine plasma anti-insulin IgG in domestic cats, and confirmed that all healthy cats had plasma anti-insulin IgG. Determining the plasma concentrations of anti-insulin IgG in cats with various pathological conditions might clarify the role of anti-insulin IgG.
Interest in xenotransplantation has increased significantly as a possible response to the critical organ shortage crisis that
exists in clinical transplantation today. The pig has been suggested to be the most suitable animal for human xenotransplantation,
however, many barriers still exist to the clinical application of xenotransplantation.
This review takes a brief look at the history of clinical xenotransplantation, the choice of organ donor, the obstacles to
xenotransplantation and attempts to overcome them. Xenografted organs will likely be used as a bridge to allotransplantation
before being used as full blown replacements for human organs. There is much progress that has been made, but still much to
do. With perseverance and good science, solutions are likely to be found.
The glycosylation enzyme alpha 1,3 galactosyltransferase, which synthesizes the carbohydrate Gal alpha 1-3Gal beta 1-4GlcNAc-R, is active in non-primate mammals, prosimians and New World monkeys, but not in Old World monkeys, apes and humans. In this study, we have cloned and sequenced the enzyme expressed in a New World monkey, determined the exact size of the stem region and assessed the minimal size of catalytically active alpha 1,3 galactosyltransferase (alpha 1,3GT). Various primer sets were used in the polymerase chain reaction to generate cDNAs which coded for forms of alpha 1,3GT with deletions at the N- or C-terminal domains. The cDNA was inserted into the expression vector pPROTA which contains the coding sequence for protein A, and subsequently transfected into COS cells. The soluble chimeric products (truncated enzyme and protein A) were harvested from the cell culture medium using IgG-Sepharose beads and assayed for enzymatic activity. As many as 67 amino acids could be truncated at the amino terminal region of the luminal portion of the enzyme without affecting its catalytic activity. Truncation of 68, 69 and 74 amino acids resulted in a 50, 75 and > 95% loss in the in vitro catalytic actively, respectively. Introduction of a frameshift mutation which is characteristic of apes and human alpha 1,3GT gene resulted in the complete loss of enzyme activity. Moreover, truncation of as few as three amino acids at the carboxyl end of alpha 1,3GT resulted in complete loss of the catalytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Employing radioimmunoinhibition assays with distinct oligosaccharides as inhibitors, this study demonstrates that the epitope recognized on mouse laminin by sera from patients with systemic sclerosis (scleroderma) is a terminal galactosyl (α1–3)‐galactose disaccharide. The reaction with this α‐digalactose was further confirmed when the sera were tested in radioimmunoassay (RIA) binding assay and in ELISA with synthetic galactose α1–3 galactosc coupled to human serum albumin. The circulating antibody appeared restricted to the IgG class and mostly to the subclass IgG3 and IgG4. Antibodies with the same specificity can be found in patients with American cutaneous leishmaniasis and Chagas disease: however, whilst in these diseases the antibody production is triggered by antigenic determinants present on the surface of the parasites, the events eliciting their appearance in systemic sclerosis are unknown.
This chapter discusses erythrocyte aging and the factors responsible for it. The mammalian erythrocyte is a unique model for the study of aging at the cellular and molecular levels, partly because it is devoid of organelles and incapable of protein synthesis. Erythrocyte aging is accompanied by the inactivation of cellular enzymes (including enzymes of antioxidative defense) and of many membrane transporters. Some of the most important functional impairments are increased hemoglobin oxygen affinity and decreased cellular deformability of aged red blood cells. There are no gross alterations in the composition of membrane proteins, but products of proteolytic degradation of Band 3 accumulate and the ratio of Band 4.1a/b increases. The latter phenomenon may be a basis for the assessment of mean red cell age in a sample. Senescent erythrocytes bind immunoglobulin G, which may take part in their elimination from the circulation. Damage of cellular constituents by reactive oxygen species may significantly contribute to the aging processes. There are various hypotheses on the mechanism of selective recognition and removal of senescent red cells invoking decreased deformability, phosphatidylserine exposure on the cell surface, and the appearance of a senescent cell antigen on molecules of the Band 3 protein (anion exchange protein).
Anti-Gal is a natural antibody, which constitutes as much as 1% of circulating IgG in humans and displays a distinct specificity for the structure Galalpha 1--> 3Gal. This glycosidic structure has been found on various tissues of many nonprimate mammals. A comparative study of the occurrence of anti-Gal versus the expression of the Galalpha 1--> 3Gal epitope was performed in primates, and a distinct evolutionary pattern was observed. Whereas anti-Gal was found to be present in Old World monkeys and apes in titers comparable to those in humans, its corresponding antigenic epitope is abundantly expressed on erythrocytes of New World monkeys. Immunostaining with anti-Gal of glycolipids from New World monkey erythrocytes indicated that the molecules to which anti-Gal binds are similar to those found in rabbit and bovine erythrocytes. These findings indicate that there is an evolutionary reciprocity between New World and Old World primates in the production of the Galalpha 1--> 3Gal structure and the antibody that recognizes it. The expression of the Galalpha 1--> 3Gal epitope was evolutionarily conserved in New World monkeys, but it was suppressed in ancestral lineages of Old World primates. The suppression of this epitope was accompanied by the production of anti-Gal. The observed in vivo binding of anti-Gal to human normal senescent and some pathologic erythrocytes implies that the Galalpha 1--> 3Gal epitope is present in man in a cryptic form.
This is a personal account of the discovery of the natural anti-Gal antibody, the most abundant natural antibody in humans, the reciprocal distribution of this antibody and its ligand the α-gal epitope in mammals and the immunological barrier this antibody has formed in porcine to human xenotransplantation. This barrier has been overcome in the recent decade with the generation of α1,3-galactosyltransferase gene-knockout pigs. However, anti-Gal continues to be relevant in medicine as it can be harnessed for various therapeutic effects. Anti-Gal converts tumor lesions injected with α-gal glycolipids into vaccines that elicit a protective anti-tumor immune response by in situ targeting of tumor cells for uptake by antigen-presenting cells. This antibody further accelerates wound and burn healing by interaction with α-gal nanoparticles applied to injured areas and induction of rapid recruitment and activation of macrophages. Anti-Gal/α-gal nanoparticle immune complexes may further induce rapid recruitment and activation of macrophages in ischemic myocardium and injured nerves, thereby inducing tissue regeneration and prevention of fibrosis.
As much as 1% of circulating IgG in man (the natural anti-Gal antibody) is directed against the α-galactosyl epitope, with the structure Galαl→3Galβl→4GlcNAc-R. The α-galactosyl epitope is abundantly expressed on cells of nonprimate mammals, prosimians, and New World monkeys. Its expression is diminished in Old World monkeys, apes, and humans. It has been previously suggested that interaction between anti-Gal and aberrantly expressed α-galactosyl epitopes on thyroid cells may contribute to the initiation of autoimmune thyroid disorders. To study this possibility, α-galactosyl epitope expression on thyroid cell membranes of normal individuals and patients with Graves' disease was assessed by a sensitive radioimmunoassay. α-Galactosyl epitopes were found both on normal and diseased thyroid cells. Whereas the concentration of these epitopes on Graves' disease thyroid membranes was somewhat higher than that observed in normal glands, the difference was not significant. The activity of the enzyme, αl-3-galactosyltransferase, which synthesizes the α-galactosyl epitope, was higher in microsomal fractions obtained from some patients as compared with healthy controls, but not significantly different. In view of the abundance of anti-Gal antibody in the circulation, it is argued that, under physiologic conditions, the interaction of this antibody with α-galactosyl epitopes does not elicit pathologic effects. However, aberrant expression of the α-galactosyl epitope may result in effective anti-Gal binding to thyroid cells (e.g., rearrangement of this structure on the cell membrane or its increased expression). Such an event may subsequently result in anti-Gal mediation of lesions which would contribute to the initiation of an autoimmune process within the thyroid gland.
Antibodies against galactosyl-1-3-galactose epitopes were characterized in normal and patient sera by radioimmunoassay binding to mouse laminin and oligosaccharide inhibition. Binding was strictly dependent on -linked galactose in a terminal position. Reduced affinities were observed for digalactoses with (1-2)-, (1-6)- and (1-4)-linkages and for the blood group B epitope, Gal1-3(Fuc1-2)Gal. Conformational models of various active and inactive oligosaccharides provided a clearer picture of the epitope requirements for the observed antibody specificity. Some antibody heterogeneity was detected by comparing individual sera and by hapten elution from a laminin adsorbent. New assays were developed with synthetic Gal1-3Gal-albumin conjugates and these were shown to be more sensitive than assays with mouse laminin. Two more ubiquitous human antibodies could be detected with Gal1-2Gal and Gal1-4Gal conjugates. They were distinct from Gal1-3Gal-specific antibodies as shown by carbohydrate inhibition. This demonstrates a considerable diversity in the recognition of -linked galactose epitopes by natural antibodies.
A substance interfering with the enzyme-linked immunosorbent assay (ELISA) for feline insulin concentration was investigated in healthy cats. An insulin-binding substance isolated from feline serum showed 2 bands at 25 and 50 kDa in SDS-PAGE, suggesting the presence of immunoglobulin G (IgG). Insulin-binding IgG from healthy cats indeed reduced insulin immunoreactivity in the ELISA for determining insulin concentration. The insulin-binding IgG was polyclonal/polyreactive and showed certain specificity, high affinity, and high binding capacity, which was evaluated by liquid-phase radioimmunoassay with Scatchard plot analysis. Epitope analysis revealed that the insulin-binding IgG showed significant binding at residues A1-5 and B20-30 of the insulin molecule. Removal of the antibodies from serum enabled the determination of serum insulin concentrations by ELISA. Our data indicated that serum from healthy cats contained substantial amounts of natural autoantibodies combined with insulin, and that the antibodies interfered with the heterologous immunoassay for serum insulin concentration.
An osmotic pulse was used to modify red blood cells (RBC) from two patients with sickle cell disease, resulting in an increased volume and decreased hemoglobin content. This treatment yielded cells which were divided into two populations, one in which RBC had markedly decreased hemoglobin concentration and another in which the cells appeared to be unmodified. Morphological sickling at low oxygen partial pressure was markedly decreased. However, there was no evidence for increased RBC lifespan when the 51Cr-labeled, modified cells were reinfused.
Mechanistic studies were conducted to examine the relationship between oxidative membrane protein damage, altered Ca2+ homeostasis, and changes in the levels of plasma membrane-bound Ca(2+)-activated neutral protease, microCANP. Alterations in the levels of plasma membrane-bound microCANP in erythrocytes and hemolysate following cumene hydroperoxide (CHP) insult were monitored using SDS-PAGE and immunoblot analyses. Free radical scavengers, antioxidant and EGTA effects on membrane-bound microCANP levels in CHP-treated cells and hemolysate were also examined. CHP (2 mM) addition to red cells caused a significant decrease/loss in intensity of numerous protein bands in the SDS-PAGE pattern, to include bands 1, 2, 2.1, 4.1, 4.2, and an approximately 60-kDa protein. N-acetylcysteine (20 mM), dithiothreitol (50 mM), and dimethylthiourea (50 mM) diminished CHP-mediated membrane protein damage; in contrast, dimethylfuran (50 mM) exacerbated CHP-mediated membrane protein damage. Dimethylsulfoxide (50 mM) was without significant effect. The free radical scavengers and antioxidants differentially affected membrane-bound microCANP levels largely in parallel with their ability to modulate membrane protein damage. Immunoblot analysis of 1 mM CHP-treated red cells revealed a time-dependent loss of membrane-bound microCANP, with a complete loss of microCANP monitored at 8 hr. Treatment of erythrocytes with CHP also resulted in concentration-dependent alterations in the level of membrane-bound microCANP: at 0.5 or 1.0 mM CHP a decreased level of membrane-bound microCANP was detected relative to control, whereas an increase in the level of bound enzyme was monitored from 2 to 4 mM CHP. CHP addition to hemolysate produced a decrease in membrane-bound microCANP levels comparable to that observed with erythrocytes; addition of the Ca2+ chelator EGTA or Calpain Inhibitor I (N-acetyl-leucyl-leucyl-leucyl-nor-leucinal) to hemolysate effectively inhibited this decrease. In contrast, treatment of erythrocytes with Ca2+ in the presence of the Ca2+ ionophore A23187 resulted in change in the SDS-PAGE protein bands and membrane-bound microCANP levels that were comparable to those produced by CHP. Inclusion of EGTA in this system prevented microCANP binding. These data provide evidence for membrane damage and concomitant dynamic alterations in membrane-bound microCANP levels in the red cell or hemolysate following oxidative insult, and show that this process can be modulated by free radical scavengers and antioxidant, simulated by treating cells with Ca2+ in the presence of ionophore, and inhibited by EGTA or Calpain Inhibitor I.
The galactose alpha 1-3 galactose (Gal alpha 1-3 Gal) residue is a carbohydrate widely distributed in many non-human mammals. Since Gal alpha 1-3 Gal residues are described on the cell surface of tumor cells, we have examined the possibility of their expression on human trophoblastic cells at different stages of placental implantation and in various pregnancy-associated conditions. Using immunohistochemical methods, Gal alpha 1-3 Gal was demonstrated on interstitial and vascular trophoblast during pregnancy. For villous trophoblast, the staining disappeared in second trimester pregnancies. The density of staining for Gal alpha 1-3 Gal was increased in highly invasive trophoblast (mole and choriocarcinoma) and decreased in poorly invasive specimens (spontaneous abortion, XO monosomia). No cells displaying Gal alpha 1-3 Gal at their surface were identified in some segments of spiral arteries from pre-eclamptic women. The anti-Gal antibody titer increased in the first trimester of pregnancy and in the sera of pre-eclamptic and eclamptic patients. These findings suggest that Gal alpha 1-3 Gal residues could be considered as markers for trophoblast invasive capacity and that the binding of maternal anti-Gal antibodies to the trophoblast could contribute to limit trophoblastic invasion and thus participate to the immunological control of implantation.
One percent of circulating IgG in humans recognizes galactose alpha 1,3 galactose residues (anti-Gal) and is synthesized in response to stimulation by enteric bacteria. In this study, we found that the prevalence of binding of anti-Gal to blood isolates is significantly higher than its binding to normal stool isolates. When anti-Gal bound onto the lipopolysaccharide of a representative blood isolate, Serratia marcescens #21, it blocked its alternative complement pathway (ACP) lysis and made the organism serum resistant. In contrast, when anti-Gal bound to the capsular polysaccharide of a serum sensitive Serratia, #7, it increased ACP killing of this strain. The mechanism of blockade of ACP lysis by anti-Gal did not involve a decrease in the number of C3 molecules deposited onto Serratia #21 or an inhibition of the binding of C3b to its LPS, nor did it change the iC3b and C3d degradation products of bound C3b or prevent membrane attack complex formation on this organism. Our findings suggest that the effect of anti-Gal on immune lysis is dependent on the bacterial outer membrane structure to which it binds. We postulate that anti-Gal may play a role in the survival of selected Enterobacteriacae in Gram-negative sepsis by blocking ACP-mediated lysis of such bacteria by the nonimmune host, and that this effect depends on where anti-Gal finds its epitope on the bacterial outer membrane.
As much as 1% of circulating IgG in man (the natural anti-Gal antibody) is directed against the alpha-galactosyl epitope, with the structure Gal alpha 1----3Gal beta 1----4GlcNAc-R. The alpha-galactosyl epitope is abundantly expressed on cells of nonprimate mammals, prosimians, and New World monkeys. Its expression is diminished in Old World monkeys, apes, and humans. It has been previously suggested that interaction between anti-Gal and aberrantly expressed alpha-galactosyl epitopes on thyroid cells may contribute to the initiation of autoimmune thyroid disorders. To study this possibility, alpha-galactosyl epitope expression on thyroid cell membranes of normal individuals and patients with Graves' disease was assessed by a sensitive radioimmunoassay. alpha-Gal-actosyl epitopes were found both on normal and diseased thyroid cells. Whereas the concentration of these epitopes on Graves' disease thyroid membranes was somewhat higher than that observed in normal glands, the difference was not significant. The activity of the enzyme, alpha 1-3-galactosyltransferase, which synthesizes the alpha-galactosyl epitope, was higher in microsomal fractions obtained from some patients as compared with healthy controls, but not significantly different. In view of the abundance of anti-Gal antibody in the circulation, it is argued that, under physiologic conditions, the interaction of this antibody with alpha-galactosyl epitopes does not elicit pathologic effects. However, aberrant expression of the alpha-galactosyl epitope may result in effective anti-Gal binding to thyroid cells (e.g., rearrangement of this structure on the cell membrane or its increased expression).(ABSTRACT TRUNCATED AT 250 WORDS)
This study demonstrates that low-density metabolically replete HbSS erythrocytes suspended in heat-inactivated autologous plasma and subjected to 15 hr of cyclical deoxygenation (under nitrogen) bind significantly increased quantities of autologous IgG as compared with oxygenated paired samples. IgG binding to the erythrocyte surface was quantified by a nonequilibrium 125-iodinated protein A binding assay and by flow cytometry. Sickle cells deoxygenated 15 hr (37 degrees C) in the presence of 2 mM calcium bound 2.2 +/- 0.2 (mean +/- SD)-fold more IgG (p less than 0.01) than oxygenated paired samples. Sickle erythrocytes deoxygenated in 0.4 mM EDTA bound 1.7 +/- 0.3 (mean +/- SD)-fold more autologous IgG than oxygenated controls (p less than 0.05). Indirect immunofluorescence assays also demonstrated that the relative levels of autologous IgG bound to sickle cells after 15 hr cyclical deoxygenation in the presence or absence of calcium was increased as compared with IgG binding by oxygenated paired samples. After 3 hr of cyclical deoxygenation in the presence of 2 mM calcium sickle erythrocytes exhibited a 40-60% increase in IgG binding, as compared with 10-20% increased IgG binding by paired samples treated in EDTA. These findings demonstrate that repeated morphologic sickling will increase the IgG binding capacity of low-density sickle cells, and suggest that sickling-associated alterations of the cell surface will produce new binding sites recognized by autologous IgG. These studies also show that the sickling-induced increase in IgG binding may be slightly enhanced by the presence of extracellular calcium.
Phenylhydrazine-mediated protein damage in human red cells has been assessed using HPLC, one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and immunoblot analysis of major membrane proteins. The association of the Ca(2+)-activated neutral protease, calpain, with membrane proteins following hydrazine insult was also examined using immunoblot analysis. HPLC amino acid analysis of red cell suspensions was employed to quantify proteolysis. Phenylhydrazine (4 mM) increased the rate of leucine, lysine, and histidine release by approximately 12-, 7-, and 5-fold, respectively. N-acetylcysteine (20 mM), dithiothreitol (50 mM), and dimethylthiourea (50 mM) decreased the rate of phenylhydrazine-stimulated amino acid release by approximately 30-50%; in contrast, the free radical scavengers and antioxidants dimethylfuran (50 mM) and dimethyl sulfoxide (50 mM) were without significant effect. The calcium chelator, EGTA (10 mM) inhibited phenylhydrazine-stimulated proteolysis by approximately 30%. Phenylhydrazine (4 mM) caused attenuation of the major membrane protein bands present in the SDS-PAGE pattern and extensive smearing of a band in the region of approximately 28 kDa. Free radical scavengers and antioxidants failed to ameliorate significantly membrane protein damage in phenylhydrazine-treated cells as judged by SDS-PAGE. Immunoblot analysis of spectrin confirmed these results. Two-dimensional SDS-PAGE of membrane proteins following phenylhydrazine treatment, however, revealed the appearance of new protein spots and a loss of existing protein spots as compared to control. Western blot analysis of membrane-associated calpain (79 kDa (proenzyme), 77- and 75-kDa forms) was also performed. Phenylhydrazine-treated red blood cells exhibited concentration- and time-dependent changes in the level of membrane-associated procalpain relative to control. The inhibitors N-acetylcysteine, dithiothreitol, dimethylthiourea, and dimethyl sulfoxide in the presence of phenylhydrazine appeared to preserve the level of procalpain in association with the membrane proteins, but only N-acetylcysteine and dithiothreitol protected the 77- and 75-kDa forms. In contrast, dimethylfuran in the presence of phenylhydrazine caused a substantial decrease in all three forms of membrane-associated calpain. In phenylhydrazine-treated hemolysate, the level of the 77- and 75-kDa forms of membrane-associated calpain was decreased relative to control. These forms were absent when EGTA (10 mM) was included in the incubation and the level of proenzyme was decreased. These data suggest that calpain is recruited to the membrane following hydrazine insult, undergoes a Ca(2+)-dependent conversion to the active forms, and may be involved in the degradation of damaged cytosolic and membrane protein(s).
A natural IgG antibody (anti-Gal) with alpha-galactosyl binding specificity has been found in large amounts (0.5 - 1.0% of serum IgG) in all individuals tested. It has been purified by affinity chromatography on a column of melibiose-Sepharose. In addition to its affinity for normal and pathological senescent human red cells, the antibody readily interacts with rabbit red blood cell (RRBC) glycolipids with alpha-galactosyl terminal residues. Two types (glycosidic linkages of 1----3 vs. 1----4) of rabbit red cells glycolipids with terminal alpha-galactosyl residues were tested for antibody binding. The antibody specifically bound to glycolipids with Gal alpha 1----3 terminal residues, and treatment of these glycolipids with alpha-galactosidase abolished binding. Hemagglutination inhibition studies with oligosaccharides of known structure also showed that the antibody binds specifically to glycoconjugates with an alpha 1----3 terminal galactose residue. Anti-Gal did not bind to a human B-active glycolipid, indicating that fucose-linked alpha 1----2 to the penultimate galactose prevents anti-Gal binding. The anti-Gal specificity for RRBC glycolipids also paralleled that of the alpha-galactosyl-specific Bandeiraea simplicifolia lectin. The possible reasons for the occurrence of this unique antibody in human serum are discussed.
Treatment of thyroglobulins from several mammalian sources (calf, sheep, pig, dog, rat, rabbit, guinea pig, and man) with alpha-galactosidase demonstrated a species-dependent occurrence of terminal alpha-D-galactosyl residues which ranged from 11 mol/mol of protein (23% of total galactose) in calf to a complete absence in man. The presence of the alpha-D-galactosyl groups resulted in a partial binding of the thyroglobulins (greater than 70% in calf and sheep) to Bandeiraea simplicifolia I-agarose, and this lectin-thyroglobulin interaction could be quantitated by a solid-phase assay utilizing 125I-labeled B. simplicifolia I. Sequential glycosidase digestions of calf thyroglobulin glycopeptides containing the complex carbohydrate unit (unit B) and characterization of oligosaccharide obtained by partial acid hydrolysis indicated that the alpha-D-galactosyl residues are located on oligosaccharide branches with an alpha-D-Gal-(1----3)-beta-D-Gal-(1----4)-D-GlcNAc sequence. While mild acid treatment of calf thyroglobulin glycopeptides yielded a disaccharide, alpha-D-Gal-(1----3)-D-Gal, and a trisaccharide, alpha-D-Gal-(1----3)-beta-D-Gal-(1----4)-D-GlcNAc, which could be resolved by B. simplicifolia I-agarose or thin-layer chromatography, similar hydrolysis of the human unit B-containing glycopeptides did not produce such components. A study of various glycopeptides indicated that the alpha-D-galactosyl residues are unevenly distributed among the multiple complex carbohydrate units of calf thyroglobulin and are preferentially located in units with a relatively low sialic acid content. During affinity chromatography on B. simplicifolia I-agarose, glycopeptides with multiple alpha-D-galactosyl groups bound more firmly to the lectin than those which contained only a single residue. In contrast to the alpha-D-galactosyl residues, beta-linked galactose of calf thyroglobulin was primarily bound in penultimate locations being susceptible to enzymatic release only after prior removal of capping sialyl and alpha-D-galactosyl groups. The isolation of N-acetyllactosamine and a beta-D-Gal----beta-D-GlcNAc----D-Man trisaccharide from partial acid hydrolysates helped to position the beta-D-galactosyl residues in the oligosaccharide branches of the complex carbohydrate units.
Hemolytic anemia in sickle disease involves both intravascular and extravascular destruction of erythrocytes. Since the latter presumably involves the reticuloendothelial system, we have examined interactions between sickle erythrocytes and macrophages. In erythrophagocytosis assays, 18.9 +/- 7.2% of human marrow macrophages ingest sickle RBCs, while only 3.1 +/- 2.1% ingest normal RBCs. This abnormality is not explained by reticulocytosis, and it is strongly dependent upon RBC density. The interaction between sickle RBCs and macrophages appears to be partly immunologic, since it is partially blocked by Fc receptor blockade. Also, admixture of sickle RBCs (pretreated with rabbit anti-human-Ig) and Fc-receptor-bearing K562 cells results in 15.6 +/- 10.6% K562-RBC rosette formation compared with only 0.5 +/- 1.2% for normal RBCs. Regarding other factors that might promote erythrophagocytosis, sickle RBCs are found to spontaneously generate twice-normal amounts of dialdehyde byproducts of lipid peroxidation ("malondialdehyde" or MDA). Peroxide or reagent-MDA treatment of normal RBCs significantly enhances their phagocytosis, and MDA is at least 50 times more potent than other aldehydes studied here. Oxidative and immunologic effects may be related, since exposure of MDA-treated RBCs to immunoglobulin-containing human sera results in a further significant enhancement of erythrophagocytosis. For comparison of different sickle patients, an adherence assay demonstrates that sickle RBCs are 1.03 to 6.85 times more adherent to macrophages than are normal RBCs, and degree of adherence correlates significantly with irreversibly sickled cell (ISC) counts and hematologic variables reflecting hemolytic rate. We conclude that propensity for RBC interaction with macrophages is likely to be a determinant of hemolytic rate in sickle disease. Pertinent mechanisms appear to involve modification of RBC membranes by dialdehyde byproducts of excessive autoxidation and the abnormal acquisition of surface immunoglobulin on sickle RBCs, although participation of other membrane defects has not been excluded. Interestingly, the data further suggest the possibility that appearance of the "senescence antigen" in old normal RBCs represents modification of the membrane by "MDA."
A new natural anti-alpha-galactosyl IgG antibody (anti-Gal) was found to be present in high titer in the serum of every normal individual studied. The antibody was isolated by affinity chromatography on a melibiose-Sepharose column. The reactivity of the antibody was assessed by its interaction with alpha-galactosyl residues on rabbit erythrocytes (RabRBC). The specificity was determined by inhibition experiments with various carbohydrates. The anti-Gal interacts with alpha-galactosyl residues, possibly on glycolipids of human RBC (HuRBC), after removal of membrane proteins by treatment with pronase. In addition, the anti-Gal bind specifically to normal and pathologically senescent HuRBC, suggesting a physiological role for this natural antibody in the aging of RBC. The ubiquitous presence of anti-Gal in high titers throughout life implies a constant antigenic stimulation. In addition to the theoretical interest in the antibody, the study of the anti-Gal reactivity seems to bear immunodiagnostic significance. Decrease in the antibody titer was found to reflect humoral immunodeficiency disorders.
IgG autoantibodies in human serum selectively bind to a glycopeptide antigen that appears on senescent and damaged cells in situ. We identified the membrane protein from which the senescent cell antigen is derived by using a phagocytosis-inhibition assay and immunoautoradiographic gel staining and electroblotting techniques. Results of the phagocytosis-inhibition assay revealed that only the purified transmembrane glycoprotein designated "band 3" and senescent cell antigen inhibited the phagocytosis of erythrocytes induced by IgG eluted from senescent erythrocytes. Purified spectrin, syndein, band 4.1, actin, glycophorin A, and intact or desialylated sialoglycoprotein periodic acid/Schiff (PAS) staining bands 1-4 containing glycophorins A, B, and C did not inhibit phagocytosis. Specific antibodies against the senescent cell antigen and erythrocyte band 3 were used to identify the membrane protein from which the senescent cell antigen is derived. Band 3-related polypeptides (MrS approximately equal to 60,000, 42,000, and 18-26,000) were identified in erythrocyte ghosts prepared in the presence of diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and EDTA by immunoautoradiography with antiband 3. Antibodies to senescent cell antigen reacted with band 3 and the same lower Mr band 3-related polypeptides. Thus, the senescent cell antigen is immunologically related to band 3.
A modified antiglobulin test, based on the high affinity between the Fc portion of the red blood cell (RBC) bound IgG and the Fc receptor on the myeloid cell K-562, was utilized for demonstration of immunoglobulins (Ig) on thalassemic RBC. Ig was found on the RBC of 73 out of 80 patients with thalassemia. The immunoglobulins on the thalassemic RBC belonged to the IgG subclass and were autoreactive. Elution studies utilizing various carbohydrates, or by thermal stripping, indicated that at least part of the IgG molecules found on the thalassemic RBC were specifically reactive with terminal galactosyl residues on the RBC membrane. IgG antibodies with similar reactivity were also demonstrated in normal human serum. These natural antigalactosyl IgG antibodies from normal sera could bind to IgG-depleted thalassemic RBC. Thalassemic RBC and normal senescent RBC were previously found to contain reduced amounts of membrane sialic acid (SA). It is suggested that the antigalactosyl IgG antibodies interact with newly exposed galactosyl residues underlying the sialic acid units. Such interaction may lead to the shortened lifespan of thalassemic RBC and may result in sequestration of senescent normal RBC by the reticuloendothelial system.
Phorbol myristate acetate (PMA) at a concentration of 0.01 microgram/ml causes an approximately threefold increase in surface area of resident, proteose-peptone-elicited, and thioglycolate-broth-elicited mouse peritoneal macrophages. Resident and proteose-peptone-elicited macrophages, cultured for 24 h in the presence of PMA, increase their pinocytic rate twofold in response to addition of PMA (0.01 microgram/ml) to the medium. Thioglycolate-broth-elicited macrophages, cultured for 24 h in the absence of PMA, immediately increase their pinocytic rate 2- to 3.5-fold in response to a single challenge with PMA (0.01 microgram/ml). Cytochalasin B, colchicine, and podophyllotoxin have only modest inhibitory effects on the basal rate of pinocytosis and on PMA-induced cellular spreading, but completely block the stimulatory effects of PMA on pinocytosis in thioglycolate-broth-elicited macrophages. Cytochalasin D markedly inhibits both basal and PMA-stimulated pinocytosis in these cells. Thus, PMA is a useful tool for studying mechanisms of macrophage spreading and for enhancing the overall rate of pinosome formation.
Heterogeneity of alpha antigen site density was demonstrated on individual human A1 and A2 type red cells by immunoelectronmicroscopy using haemocyanin-conjugated antiglobulin. Examination of red cells fractionated on a Percoll density gradient showed that the heterogeneity was not age-related. The presence of autologous immunoglobulin on senescent red cells was also confirmed by the same technique.
A subhuman primate model was developed to ascertain whether or not platelet heterogeneity could be explained by aging in the peripheral circulation. Density-dependent platelet cohorts, postulated to represent cells of different ages, were isolated on isosmolar arabinogalactan gradients and labeled with radiochromium. Mean platelet lifespan was measured for the different density cohorts, and simultaneous sequential density distribution analysis was performed to follow changes in cell density during aging. The average mean lifespan of light platelets was 74.6 hr, compared to 313.6 hr for heavy platelets. After injection, labeled light platelets were recovered only in the gradient light region, in contrast to labeled heavy platelets, which were initially restricted to the dense region and progressively migrated to the light region during their lifespan. This study supports the hypothesis that platelet age in unstressed primates correlates with cell density and provides a rationale for the use of "age-dependent" markers to estimate platelet turnover rates.
Physiological or experimental decrease in sialic acid (SA) content on the red blood cell (RBC) membrane is believed to play an important role in the recognition of these cells by macrophages. Since there is a 20-30% decrease in the SA content on the membrane of thalassaemic RBC, the interaction between macrophages and these RBC was studied in vitro. Using mouse peritoneal macrophages, it was found that these macrophages 'recognize' and phagocytize thalassaemic RBC while RBC from normal donors are hardly phagocytized. The average level of phagocytosis of thalassaemic RBC from splenectomized patients was found to be 22-fold higher than that of RBC from normal donors. The phagocytized cells consisted of both mature and nucleated RBC. Mouse peritoneal macrophages seem to be a useful in vitro system for the study of the accelerated sequestration and shortened life span of thalassaemic RBC.
Mechanisms by which macrophages discriminate between "mature-self" and "senescent-self" were investigated using the human red blood cell (RBC) system as a model. Conditions simulating those encountered in situ were adhered to as closely as possible by using short term culture techniques and incubating with autologous macrophages and immunoglobulins (Ig). It was found that RBC aged in vitro were phagocytized when they were incubated in pooled, normal human IgG, allogeneic Ig, autologous IgG or Ig, washed with medium, and then incubated with autologous macrophages. RBC treated in the same way but incubated in IgM or Iga, Ig-depleter serum, or Medium 199 alone were not phagocytized. This indicates that Ig is required for phagocytosis and suggests that the Ig which attaches to RBC is IgG. When freshly drawn RBC were separated into young (Y) and old (O) RBC according to density and incubated with autologous macrophages, less than 5% of the Y-RBC were phagocytized, whereas greater than 30% of the O-RBC were phagocytized, independent of whether the final incubations were performed in medium without serum (Y-RBC, 5 +/- 2% phagocytized; O-RBC, 33 +/- 1.5%), or autologous Ig-depleted serum (Y-RBC, 2 +/- 2.5%; O-RBC, 51 +/- 17%), or whole serum (Y-RBC, 0%; O-RBC, 43 +/- 5%). This indicates that (1) the Ig is attached in situ to the old RBC, and (2) that phagocytic recognition is not inhibited by other serum components. Scanning electron microscopy, employing labeled anti-IgG, IgM, and IgA reagents, revealed that Y-RBC had essentially no Ig on their surface, whereas old RBC had IgG on their surface. These findings indicate that IgG attaches in situ to senescent human RBC, making them vulnerable to phagocytosis by macrophages.
The existence of hemolysis in sickle cell disease has been documented by both indirect and direct methods. The existence of bone-marrow erythroid hyperplasia, reticulocytosis, indirect hyperbilirubinemia, and elevations of plasma hemoglobin and serum lactic acid dehydrogenase values show hemolytic disease. Direct studies of erythrocyte survival, including the Ashby differential agglutination technique as well as isotopic methods have all shown a markedly decreased red blood cell survival in the range of 10 to 30 days mean cell life-span. Data obtained by means of endogenous production of carbon monoxide have shown that the mean rate of heme catabolism is approximately six times normal but varies from 3 to 14 times normal in individual patients. These data document, by a relatively new technique, the consistent presence of a severe hemolytic process in sickle cell anemia.
Relatively heavy as well as relatively light human erythrocytes, obtained by ultracentrifugation of normal blood in dextran solutions of different density, were labelled with 51Cr and re-injected into the same normal individual. By measuring the radioactivity of the blood after different intervals, the moment was determined at which radioactivity in the bloodstream had diminished to 50% (51Cr T1/2). The 51Cr T1/2 of the relatively heavy erythrocytes proved to be shorter, and that of the relatively light erythrocytes proved to be longer than that of unfractioned samples submitted to similar processing. In addition the relaively heavy fractions showed a highly variable, early loss during the first 24 hours following re-injection.
Rabbit red blood cells were separated in groups of different specific gravity by ultracentrifugation in a discontinuous gradient of isosmotic bovine serum albumin. These fractions were easily separated without mixing by slicing the tube through the albumin layers. The red cells behaved as osmometers, and their specific gravity changed with deviation of the medium from isotonicity. Reproducible results could only be obtained if the osmolality was rigidly controlled. The relationship between red cell specific gravity and age of the cells was evaluated by cohort labeling with glycine-2-C14 and Fe59. The results obtained with Fe59 were confused by the reutilization of the label. The results obtained with glycine-2-C14 demonstrated a progressive increase of the red cell specific gravity with age. A comparison was made between ultracentrifugation with and without gradient; the use of the gradient allows density equilibrium to be reached and much sharper age-dependent fractionation. A mathematical model has been elaborated, taking into account the random loss of cells as well as the broad normal distribution of cells in regard to specific gravity, to describe the distribution of red cells of different age at various levels in the gradient. The cell volume was found to be decreased in older cells, but hemoglobin content was unchanged; thus, hemoglobin concentration was found to be increased.
Erythrocyte-associated IgG was measured on washed red cells from 26 children with P. falciparum parasitaemia using an immunoradiometric assay. The values obtained were compared with 15 age-matched controls living in the same area who did not have malaria. The amount of red cell-associated IgG was related to the haemoglobin concentration, degree of parasitaemia and serum immunoglobulin level. The mean red cell-associated immunoglobulin for patients with malaria was 629 molecules per red cell (range 215-1770) which was significantly higher than the control group, who had a mean of 395 molecules per red cell (range 190-930). A statistically significant correlation was established between red cell-associated IgG and the degree of anaemia in the infected patients. In contrast, there was no relationship between the red cell-associated IgG and the degree of parasitaemia or the serum immunoglobulin level. These observations support the hypothesis that an immunological mechanism could be involved in the anaemia associated with malarial infections.
Macrophages secrete a variety of biologically active substances into their local milieu, including proteins, lipids, nucleotide metabolites, and oxygen metabolites. To date, more than 50 substances secreted by macrophages have been reported: enzymes; enzyme inhibitors; plasma proteins such as complement components, coagulation factors, and apolipoprotein E; factors that regulate the functions of other cells such as interferon, interleukin 1, mitogens, and angiogenesis factor; and low molecular weight substances such as reactive metabolites of oxygen and derivatives of arachidonic acids. Macrophage-derived products are probably important in the local environment, and they are believed to be important in the physiological and pathological functions of macrophages in inflammation, tissue repair, lipoprotein metabolism, acute phase response, and in microbicidal, antiviral, tumoricidal, and immunoregulatory activities; however, macrophages may not be the sole source for the secretion of some of these products. The secretion of these products is intricately regulated, developmentally and environmentally.
The erythrocyte-antibody rosetting antiglobulin test was used to determine differences in the expression of various Rh antigens on homozygous and heterozygous Rh positive red blood cells. The rosetting antiglobulin test was able to detect the relative difference in expression of C, c, E and e on homo- and heterozygous red blood cells. Based on these findings, the test was used and found suitable for determining the genotypes of various D positive red blood cells.
A glucose-dependent hemagglutinin, present in the serum of an apparently healthy donor, is shown to be a polyclonal IgM immunoglobulin with specificity for beta-D-glucopyranose configuration. The antibody did not agglutinate erythrocytes coated with hexoses differing from glucose at C2, C3, or C4 positions or lacking the primary alcohol group at C6 position. The hemagglutination reaction, quantitated in a continuous flow system, was inhibited by the addition of 6-deoxyglucose, 5-thioglucose, 2-deoxyglucose, 2-aminoglucose or 3-0 methylglucose. D-glucose, in beta- but not in alpha-configuration, attached by a glycosidic bond to another glucose or to an aglycone, was also inhibitory. A cross-reactivity was demonstrated between glucose and 6-deoxyglucose by antibody absorption and elution techniques. The donor's serum contained independent antibodies that reacted with erythrocytes coated with melibiose, gentiobiose, cellobiose, or N-acetylmannosamine. Further investigation revealed that antibodies are present in sera of all normal adults against erythrocytes coated with melibiose and N-acetylmannosamine and with high frequency against erythrocytes coated with gentiobiose, L-rhamnose, cellobiose, D-mannose, lactose, or D-galactose.
Immunoglobulin G (IgG) of healthy human blood donors and IgG from pooled sera (Sandoglobulin) contain natural (auto)antibodies to band 3 protein, the major integral membrane protein of human red blood cells. Affinity-purified and 125I-iodinated anti-band 3 antibodies bound specifically to band 3 protein on immunoblots from membrane proteins in the presence of unlabeled, absorbed IgG. Purified (auto)antibodies also bound nonspecifically to band 4.2 and weakly to band 5 and 6, when assayed with second antibody and 125I-iodinated protein A. The antibodies were directed to regions of band 3 protein that were cryptic and in part exoplasmic but with a low accessibility to surface modifications. The antigenic sites were located within the 65K, but not the 38K-dalton chymotryptic fragment of band 3 protein. Antigenic band 3 protein was equally present in membranes of young and senescent red cells. Hence, if these antibodies were involved in recognizing a few exoplasmic sites of band 3 protein on senescent red cells, antigen exposure would require alterations in band 3 accessibility (conformation, topology) rather than an enzymatic generation of antigenic sites.
Normal human sera contain heterophile hemagglutinins to rabbit erythrocytes which are different from anti-B isoantibody and other heterophile antibodies such as Hanganutziu-Deicher antibody or Paul-Bunnell antibody. The antigen to this antibody was purified from rabbit erythrocyte stroma, and identified as pentaglycosyl ceramide, Gal(alpha 1-3)Gal(beta 1-4)GlcNAc(beta 1-3)Gal(beta 1-4)Glc-Cer.
A family of glycopeptides that contain nonreducing terminal alpha-D-galactosyl residues has been isolated from Pronase digests of delipidated Ehrlich ascites tumor cells. The glycopeptides, which comprise 17.2% of the total plasma membrane hexose, have an average molecular weight of 7500 and are precipitated by Griffonia simplicifolia B4 isolectin, wheat germ agglutinin, and Ricinus communis lectin. Exo- and endoglycosidase digestion, periodate oxidation, permethylation analysis, and lectin reactivity provided evidence for a tentative carbohydrate structure for the glycopeptide mixture. The glycopeptides possess tetraantennary branched structures containing a trimannosyl core N-glycosidically linked via an N,N'-diacetylchitobiosyl unit to an asparagine residue. Each branch contained repeating leads to 3)-beta-D-Galp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to units resulting in a keratan-like structure, terminated with alpha-D-Galp-(1 leads to 3)-[alpha-D-Galp-(1 leads to 6)]-beta-D-Galp-units. The variation in the molecular weight observed for the glycopeptide mixture can be attributed to the variable amounts of leads to 3)-beta-D-Galp-(1 leads to 4)-beta-D-GlcNAcp-(1 leads to units found in the branch chains.
We have used the complement-fixing antibody consumption ( CFAC ) test to detect small concentrations of IgG on red blood cells from patients with hemolytic anemias that are not thought to be caused by an immune mechanism. Although patients with hereditary spherocytosis, pyruvate kinase deficiency, and mechanical hemolytic anemias generally had normal concentrations of IgG bound to their red cells (less than 25 molecules IgG per red cell), we found that 39/62 (63%) patients with sickle cell anemia had elevated values. These 39 patients had a mean of 195 and a maximum of 890 molecules of IgG per red cell. None of the patients had been transfused within the previous 90 days, and some had never been transfused. Direct antiglobulin tests were positive in only two instances and autoantibodies were not found in the serum of any patient. However, eluates from the red cells of 6 of 23 patients demonstrated antibody activity against all of a panel of normal red cells by the indirect antiglobulin test. There was no correlation between the number of IgG molecules on patients' red cells and the severity of their anemia, the incidence of painful sickle cell crises, the reticulocyte count, or with blood transfusion history. We conclude that further study of immunohematologic abnormalities in patients with sickle cell anemia is warranted, especially in view of previous reports in this population of patients with red cell autoantibodies, autoimmune hemolytic anemia, hemolytic transfusion reactions without detectable alloantibodies, and an association of some episodes of pain crises with immunologically mediated red cell destruction.
Autologous membrane-bound IgG was isolated from a subpopulation of human red blood cells (RBC) with specific density greater than 1.110, by affinity chromatography of purified RBC membrane glycoprotein preparations using immobilized wheat germ agglutinin and immobilized anti-human immunoglobulin (Ig) as immunoabsorbents. The Ig-containing population thus obtained, when further separated by chromatography on Sephadex G-200 in the presence of chaotropic agents, yielded four peaks (Ia, Ib, II, and III). Double immunodiffusion revealed the presence of Ig in the first three peaks (IgM in peak Ia, IgA in Ib, and IgG in II) but not in peak III. Peak III was precipitated by the Ig-containing peaks (Ia, Ib, and II) in immunodiffusion assays, suggesting that the antigenic membrane determinants responsible for the binding of autologous Ig to senescent human RBC were contained in this peak (III). Peaks Ia, Ib and II precipitate purified asialoglycophorin; peak III was reactive with purified autoantibodies directed against asialoglycophorin. These results suggest that an age-related antigenic determinant(s) present on senescent human RBC is exposed by desialylation of the major sialoglycoprotein component of the RBC membrane.
Direct antiglobulin (Coombs') tests of erythrocyte (RBC) subpopulations confirmed the presence of membrane-bound immunoglobulin G (IgG) on old (density greater than 1.110) human RBCs but not on the young (density less than 1.110) RBCs. After thermal elution of the bound IgG, this Coombs' reaction was negative, but incubation of thermally eluted IgG (He-IgG) with heat-treated RBCs induced a positive antiglobulin test. A positive direct antiglobulin reaction was also obtained after incubation of heat-treated RBCs with anti-T antibody. Similar results were obtained when young RBCs treated with Vibrio cholerae neuraminidase (VCN) were incubated with anti-T or with IgG eluted by heat from old RBCs. Nevertheless, pre-absorption of heat-eluted IgG with T and/or Tn antigen, did not prevent it from binding to either heat-treated old or VCN-treated young RBCs as assessed by the antiglobulin consumption assay. Pre-treatment of either VCN-treated young or heat-treated old RBCs with anti-T and/or anti-Tn antibodies had no significant effect on the binding of radiolabeled He-IgG (eluted from old RBCs). The results indicate that even though desialylation of the erythrocyte membrane is required for binding of both anti-T-Tn and He-IgG, the specificity and consequently the RBC binding sites for He-IgG and anti-T seem to be different.
Erythrocyte antibody (EA) rosette formation with the Fc-receptor on the K-562 erythro-myeloid cell line was employed for the detection of subagglutinating amounts of Ig molecules bound to red cells. The sensitivity of this method exceeds that of the conventional direct and indirect antiglobulin tests without any alteration of the incubation media or pretreatment of red cells. The increased sensitivity did not diminish the specificity of the test, which can detect IgG, IgM and complement as well. This method may demonstrate the presence of antibodies on red cells in patients with suspected autoimmune haemolytic anaemia and negative antiglobulin test.
Human sera were screened by passive haemagglutination for antibodies to various sugars. A high incidence of antibodies to melibiose was observed. There were also a few antibodies to other sugars and to dextran.
In clinical studies, frequent hepatic dysfunction associated with crises in sickle cell disease has been noted, but whether irreversible morphologic changes arise from these transient episodes is uncertain. We studied 70 patients with sickle cell disease (57 SS, 12 SC and one S-thalassemia (S-thal) hemoglobin) autopsied at The Johns Hopkins Hospital. They ranged in age from five months to 75 years (average 21 years) and 35 (50 percent) were female, In 64 patients (91 percent), livers were enlarged and had distention of Kupffer cells with phagocytized sickled red cells; this was massive in 10. In 19 patients (27 percent) the sinusoids were markedly distended with sickled red cells and appeared obstructed. Focal parenchymal necroses were present in 24 patients (34 percent) and were explained in 12, eight by cardiac dysfunction and four by sepsis. Reparative changes, portal fibrosis and regenerative nodules were each found in 14 patients (20 percent), only one of whom had a known history of viral hepatitis despite the frequency of transfusions. Cirrhosis of unknown cause was present in seven patients and cardiac cirrhosis in one. Cirrhosis with hemochromatosis was present in three patients and 30 others had parenchymal iron accumulation. Thus, unexplained hepatic necroses, portal fibrosis, regenerative nodules and cirrhosis were frequently encountered in these patients. This spectrum of liver disease appears to be best understood as a consequence of recurrent vascular obstruction, necrosis and repair arising as a component of sickle cell disease.
In 1927, in a classic paper, Hahn and Gillespie 19 clearly defined the basis of the anomalous physical behavior of the sickle cell as an abnormality of its hemoglobin when in the deoxygenated state and pointed to the affected cells as the primary cause of the characteristic hemolytic anemia. From microscopic observations of drops of blood suspended in a gas chamber and from the results of the removal of a large spleen from a child with sicklemia, they were led to make the following statement: Sickle cell formation is a reversible phenomenon depending on the free or combined state of the hemoglobin of the susceptible corpuscles. When the hemoglobin is in the combined state, the discoid form is stable; when in the uncombined state, the distorted form is stable. The behavior of the susceptible corpuscles toward oxygen asphyxia thus constitutes a special application of a hypothesis correlating contour with
Hemolysis in sickle cell disease Studies on the destruction of red blood cells. X. The biophysics and biology of sickle cell disease
- E A Besinger
- P N Gillette
Besinger, E. A., and P. N. Gillette. 1974. Hemolysis in sickle cell disease. Arch. Intern. Med. 133:624-631. 2. Harris, J. W., H. H. Brewster, H. H. Ham, and W. B. Castle. 1956. Studies on the destruction of red blood cells. X. The biophysics and biology of sickle cell disease. Arch. Intern. Med. 97:145-168.
The liver in sickle cell disease: A clinicopathologic study of 70 patients Mechanism of removal of senescent cells by human macrophages in situ Mechanisms for removal of senescent human erythrocytes from circulation
- T W Bauer
- W Moore
- G M Hutchins
- M M B Kay
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