Factor H interferes with the adhesion of sickle red cells to vascular endothelium: a novel disease-modulating molecule.

Sickle cell disease is an autosomal recessive genetic red cell disorder with a worldwide distribution. Growing evidence suggests a possible involvement of complement activation in the severity of clinical complications of sickle cell disease. In this study we found activation of the alternative complement pathway with microvascular deposition of C5b-9 on skin biopsies from patients with sickle cell disease. There was also deposition of C3b on sickle red cell membranes, which is promoted locally by the exposure of phosphatidylserine. In addition, we showed for the first time a peculiar "stop-and-go" motion of sickle cell red blood cells on tumor factor-α-activated vascular endothelial surfaces. Using the C3b/iC3b binding plasma protein factor Has an inhibitor of C3b cell-cell interactions, we found that factor H and its domains 19-20 prevent the adhesion of sickle red cells to the endothelium, normalizing speed transition times of red cells. We documented that factor H acts by preventing the adhesion of sickle red cells to P-selectin and/or the Mac-1 receptor (CD11b/CD18), supporting the activation of the alternative pathway of complement as an additional mechanism in the pathogenesis of acute sickle cell related vaso-occlusive crises. Our data provide a rationale for further investigation of the potential contribution of factor H and other modulators of the alternative complement pathway with potential implications for the treatment of sickle cell disease.

Sickle cells produce functional immune modulators and cytotoxics.

Sickle erythrocytes' (SSRBCs) unique physical adaptation to hypoxic conditions renders them able to home to hypoxic tumor niches in vivo, shut down tumor blood flow and induce tumoricidal responses. SSRBCs are also useful vehicles for transport of encapsulated drugs and oncolytic virus into hypoxic tumors with enhanced anti-tumor effects. In search of additional modes for arming sickle cells with cytotoxics, we turned to a lentiviral ß-globin vector with optimized Locus Control Region/ß-globin coding region/promoter/enhancers. We partially replaced the ß-globin coding region of this vector with genes encoding T cell cytolytics, perforin and granzyme or immune modulating superantigens SEG and SEI. These modified vectors efficiently transduced Sca+ ckit- Lin- hematopoietic stem cells (HSCs) from humanized sickle cell knockin mice. Irradiated mice reconstituted with these HSCs displayed robust expression of transgenic RNAs and proteins in host sickle cells that was sustained for more than 10 months. SSRBCs from reconstituted mice harboring SEG/SEI transgenes induced robust proliferation and a prototypical superantigen-induced cytokine reaction when exposed to human CD4+/CD8+ cells. The ß-globin lentiviral vector therefore produces a high level of functional, erythroid-specific immune modulators and cytotoxics that circulate without toxicity. Coupled with their unique ability to target and occlude hypoxic tumor vessels these armed SSRBCs constitute a potentially useful tool for treatment of solid tumors.

Human natural anti-alpha-galactosyl IgG. II. The specific recognition of alpha (1----3)-linked galactose residues.

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.

Sickle cell disease: a review.

The substitution of one amino acid in the hemoglobin molecule results in sickle hemoglobin. As a result, RBCs sickle in low oxygen states causing occlusion of blood vessels, increased viscosity, and inflammation. These RBCs are prematurely removed from the circulation, resulting in a chronic hemolytic anemia. With newborn screening and early treatment, the death rate among children with SCD has declined. In addition, a variety of treatments are being introduced to help manage the various manifestations of disease. Transfusion, simple or exchange, is a mainstay of therapy, since it reduces the amount of Hgb S in circulation and suppresses erythropoiesis. Transfusion is indicated for symptomatic anemia and specifically to prevent stroke (first or recurrent), during acute stroke, and for acute chest syndrome. Unfortunately, transfusion carries risks for infectious disease transmission, as well as immunologic and inflammatory sequelae. For patients with SCD who may be chronically transfused, iron overload occurs frequently. In addition, due to differences in RBC antigens between donors and recipients, these patients are at increased risk for development of RBC alloantibodies, which can complicate further transfusion. It is, therefore, important to prevent alloimmunization by transfusing leukoreduced RBCs that match the patient for the C, E, and K1 antigens. Human progenitor cell (from bone marrow, peripheral blood stem cells, or umbilical blood) transplant can cure the disease, and is used for patients with severe disease for whom conventional therapy may not be effective.

Alteration of human erythrocyte membrane properties by complement fixation.

Erythrocyte survival studies of complement-coated radiolabeled erythrocytes have shown rapid removal of these cells from the peripheral blood with a return of these cells into the circulation within a few hours. We studied complement-coated human erythrocytes and measured surface charge and deformability, two parameters believed to be important in erythrocyte survival. Erythrocytes were coated with complement by two in vitro techniques: the addition of (a) low ionic strength sucrose, and (b) IgM cold agglutinins. Erythrocytes obtained from three patients with cold agglutinin disease were used as a source of in vivo complement-coated cells. No difference was found in surface charge as measured by electrophoretic mobility between erythrocytes from normal subjects and complement-coated erythrocytes from any of the three sources. When deformability was measured by filtration through 3-mum polycarbonate sieves, marked decreases in deformability were found in complement-coated erythrocytes. The filtration returned toward control levels by incubating the complement-coated erythrocytes in serum for 1 h and correlated with decreases in immune adherence. Using screen filtration pressure as a measure of deformability, a positive correlation between number of C3 molecules per erythrocyte and decreased deformability was found. C3b appeared responsible for the decreased deformability of the erythrocytes, since conversion of C3b to C3d resulted in a return of deformability toward normal. The data suggested that the sequestration of complement-coated human erythrocytes in the microvasculature can be explained in part by decreased deformability and changes in immune adherence.

Excessive binding of natural anti-alpha-galactosyl immunoglobin G to sickle erythrocytes may contribute to extravascular cell destruction.

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.

Potentiated adherence of sickle erythrocytes to endothelium infected by virus.

Systemic viral infection is a known precipitant of vasocclusive crisis in sickle patients, but the mechanism underlying this clinical observation is unknown. In the present studies, human umbilical vein endothelial cells were infected with Herpes simplex virus type 1 (HSV) to model systemic viral disease. The already abnormal adherence of sickle erythrocytes to control endothelium is enhanced 1.8 +/- 0.4-fold to HSV-infected endothelium (P less than 0.001). This component of potentiated adherence is eliminated by maneuvers that block Fc receptors, it is prevented by tunicamycin, and it is not seen using a mutant HSV that is unable to express the Fc receptor glycoprotein. Thus, the incremental adherence seen here occurs due to expression of Fc receptor activity on HSV-infected endothelium and the consequent recognition of abnormal amounts of IgG on sickle erythrocytes. We conclude that systemic viral infection potentially can induce a novel mechanism for enhancement of erythrocyte adherence to endothelium and that this may increase the likelihood of vasocclusion during viral infection.

Diagnostic advances in defining erythropoietic abnormalities and red blood cell diseases.

The majority of clinical applications of flow cytometry begin with various approaches to remove red blood cells (RBCs) from the clinical sample. However, multiparameter cytometry has and will continue to contribute much to the understanding of the pathophysiology and diagnostic accuracy in the clinical evaluation of human diseases affecting erythroid cells. This review summarizes the diagnostic advances relating to erythroid cells in the areas of immunohematology, laboratory hematology, and infectious disease with particular emphasis on medical evaluation of the anemic patient and fetomaternal hemorrhage. Semin Hematol 38:148-159.

Delayed immune response in chorea-amyotrophy with spherocytosis.

Progressive CSF lymphocytic pleocytosis and intrathecal IgG-synthesis occurred late in familial amyotrophy, neuropathy, chorea, and dementia with spherocytosis. Immunoblotting showed a serum and CSF antibody apparently directed against glial fibrillary acidic protein. A secondary autoimmune response was probably triggered during the evolution of the neurodegenerative process.

Spurious macrocytosis, a common clue to erythrocyte cold agglutinins.

In four patients with cold agglutinins, erythrocyte mean corpuscular volume was increased. Erythrocyte volume histograms showed that in each case macrocytosis was due entirely to doublet erythrocytes counted as single cells. For two of the four patients, macrocytosis was reported intermittently, when the blood had been allowed to cool to room temperature during laboratory processing. For these two patients, macrocytosis due to doublet erythrocytes could be elicited in vitro proportionate to the degree of cooling of the blood and could be abolished to rewarming to 37 C. Two other patients had macrocytosis constantly during the acute illness. Otherwise unexplained macrocytosis may reflect cold agglutinin activity without overt hemolysis, particularly if the macrocytosis is intermittent.

Membrane channel protein abnormalities and autoantibodies in neurological disease.

Immunological analogues of band 3, the anion transporter of the human erythrocyte, have been identified in all cells, including both isolated neurons and neurons of the central nervous system. We hypothesized that the anion channel is altered in neurological disease associated with choreiform movements because gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in mammalian brain, binds to its receptor and opens an integral membrane chloride channel. In order to examine this hypothesis, we studied a family with a serious, progressive, genetic neurologic disorder with acanthocytosis (choreoacanthocytosis) that resembles Huntington's chorea. We selected choreoacanthocytosis because erythrocytes, which are readily obtained, are affected in this disease as well as the central nervous system. Biochemical studies of erythrocytes from the proposita, mother, and brother revealed that sulfate transport Vmax was increased, and glucose efflux was decreased. Erythrocytes exhibited immunological changes indicative of cellular aging/transporter damage. In addition, transporter reactive antibodies were present. This is the first evidence for abnormalities of membrane transport in this neurologic disorder.

A multiplicity of erythrocyte glycolipids of the neolacto series revealed by immuno-thin-layer chromatography with monoclonal anti-I and anti-i antibodies.

The thin-layer-chromatography immunostaining procedure was applied to human erythrocyte glycolipids using monoclonal anti-i and anti-I antibodies which are directed against epitopes on linear and branched carbohydrate chains of the neolacto (poly-N-acetyllactosamine) series. An examination of native and mild-acid-treated glycolipids from normal adult (I(adult) antigen type), neonatal (i(cord)), and I-antigen-deficient adult (i(adult)) erythrocytes enabled certain structural inferences to be made as follows: (a) cells of both I and i phenotypes contain a multiplicity of glycolipids of the neolacto series whose backbones consist of 8 or more sugar residues; (b) the octasaccharide backbones are predominantly linear in cells of i phenotype and branched in those of I type; and (c) more complex glycolipids having decasaccharide and larger backbones with both linear and branched sequences occur in erythrocytes of both phenotypes.

[The role of autoimmune hemolytic processes in echinocytic erythrocyte transformation in malignant growths]. / Rol' autoimmunnykh gemoliticheskikh protsessov v ékhinotsitarnoi transformatsii éritrotsitov pri zlokachestvennom roste.

No direct dependence between the surface change of erythrocyte, echinocytosis, and the activity of lymphocytes producing hemolysins in experimental carcinogenesis was revealed. It is supposed that the echinocytic transformation of erythrocytes under these conditions may be due to the influence of tumour metabolites or other factors, for example, viruses).

Sickle erythrocytes target cytotoxics to hypoxic tumor microvessels and potentiate a tumoricidal response.

Resistance of hypoxic solid tumor niches to chemotherapy and radiotherapy remains a major scientific challenge that calls for conceptually new approaches. Here we exploit a hitherto unrecognized ability of sickled erythrocytes (SSRBCs) but not normal RBCs (NLRBCs) to selectively target hypoxic tumor vascular microenviroment and induce diffuse vaso-occlusion. Within minutes after injection SSRBCs, but not NLRBCs, home and adhere to hypoxic 4T1 tumor vasculature with hemoglobin saturation levels at or below 10% that are distributed over 70% of the tumor space. The bound SSRBCs thereupon form microaggregates that obstruct/occlude up to 88% of tumor microvessels. Importantly, SSRBCs, but not normal RBCs, combined with exogenous prooxidant zinc protoporphyrin (ZnPP) induce a potent tumoricidal response via a mutual potentiating mechanism. In a clonogenic tumor cell survival assay, SSRBC surrogate hemin, along with H(2)O(2) and ZnPP demonstrate a similar mutual potentiation and tumoricidal effect. In contrast to existing treatments directed only to the hypoxic tumor cell, the present approach targets the hypoxic tumor vascular environment and induces injury to both tumor microvessels and tumor cells using intrinsic SSRBC-derived oxidants and locally generated ROS. Thus, the SSRBC appears to be a potent new tool for treatment of hypoxic solid tumors, which are notable for their resistance to existing cancer treatments.

Parietal cell antibody identified by ELISA is superior to immunofluorescence, rises with age and is associated with intrinsic factor antibody.

Parietal cell antibody is a marker for autoimmune gastritis. With identification of gastric H/K ATPase as its molecular target, ELISAs have been introduced. We compared performance of ELISA with immunofluorescence in a retrospective and prospective sera set and correlated the results with intrinsic factor antibody. In 138 retrospective sera selected for positivity or negativity for intrinsic factor antibody, 87 reacted with gastric H/K ATPase by Euroimm ELISA but only 62 reacted by immunofluorescencence.. Similar results were obtained with Inova ELISA with 78 positives that were also positive by Euroimm ELISA. In 161 prospective sera, 29 sera tested positive by ELISA compared to 24 by immunofluorescence. ELISA positive but immunofluoresnce negative sera are bona fide positives because a representative set of 16 sera reacted with both 95kD α and 60-90kDß subunits of gastric H/K ATPase. ELISA values rose with age regardless of whether immunofluorescence tests were positive or negative. Of 53 sera containing antibody to intrinsic factor, 46/53 (87%) reacted to gastric H/K ATPase by ELISA. Taken together, the data indicates an enhanced detection rate by ELISA over immunofluorescence and validates it as a robust diagnostic assay for parietal cell antibody. As parietal cell antibody marks asymptomatic autoimmune gastritis that may progress to end stage gastric atrophy and haematological complications, and as autoimmune gastritis is associated with autoimmune thyroiditic and type 1 diabetes mellitus, early detection of parietal cell antibody by a sensitive ELISA will enable early follow-up of at risk subjects.

Non-opsonising aggregates of IgG and complement in haemoglobin C erythrocytes.

Haemoglobin C (HbC) differs from normal HbA by a lysine for glutamate substitution at position 6 of beta-globin. Heterozygous AC and homozygous CC phenotypes are associated with shortened erythrocyte life spans and mild anaemia. AC and CC erythrocytes contain elevated amounts of membrane-associated haemichromes, band 3 clusters, and immunoglobulin G (IgG) in vivo. These findings led us to investigate whether AC and CC erythrocytes might expose elevated levels of IgG and complement, two opsonins that have been implicated in the phagocytic clearance of senescent and sickle erythrocytes. Surprisingly, we found IgG, complement, and other plasma proteins co-localised in aggregates beneath the membrane of circulating AC and CC erythrocytes. These observations, and our finding of similar aggregates in erythrocytes heterozygous or homozygous for haemoglobin S (sickle-cell haemoglobin), suggest that the vast majority of membrane-associated IgG and complement detected in these abnormal erythrocytes is intracellular and does not contribute to the eventual opsonic clearance of these cells. Phagocytosis studies with macrophages provide evidence in support of this suggestion. Studies of erythrocyte clearance that involve the detection of membrane-associated IgG and complement as putative opsonins should investigate the possibility that these plasma proteins reside in the erythrocyte interior, and not on the cell surface.