Biomedical subjects

M J Branks

Publications and source records attributed to M J Branks.

7 recordsLinked to original sources

PubMed1990

IgG red blood cell autoantibodies in autoimmune hemolytic anemia bind to epitopes on red blood cell membrane band 3 glycoprotein.

Red blood cell (RBC) autoantibodies from patients with IgG warm-type autoimmune hemolytic anemia were labeled with iodine 125 and their RBC binding behavior characterized. Epitope-bearing RBC membrane polypeptides were identified after autoantibody immunoprecipitation of labeled membranes and immunoblotting. Immunoaffinity isolation of labeled membrane proteins with 12 different IgG hemolytic autoantibodies with protein A-agarose revealed a major polypeptide at Mr 95 to 110 kd, which coelectrophoresed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with a membrane component isolated with sheep IgG anti-band 3. Immunoprecipitation studies with chymotrypsinized RBCs resulted in the recovery of two labeled membrane polypeptides with molecular weights characteristically resulting from the chymotryptic fragmentation of band 3. Immunoblotting with sheep IgG anti-band 3 of the immunoprecipitated polypeptides confirmed that hemolytic autoantibody binding led to recovery of band 3 or its fragments. Two 125I-labeled IgG hemolytic autoantibodies showed binding behavior consistent with epitope localization on band 3. The labeled RBC autoantibodies bound immunospecifically to all types of human RBC tested, including those of rare Rh type (Rh-null, D--) at a site density of approximately 10(6) per RBC. The 125I-IgG in two labeled autoantibodies was 84% and 92% adsorbable by human and higher nonhuman primate RBCs. Antigen-negative animal RBC bound less than 10% (dog, 2.6%; rhesus monkey, 7.4%), consistent with immunospecific RBC binding. IgG-1 was the major subclass in five autoantibodies tested; one of six fixed complement; and autoantibody IgG appeared polyclonal by isoelectric focusing. We conclude that IgG eluted from RBCs of patients with autoimmune hemolytic anemia consists predominantly of a single totally RBC-adsorbable antibody population that binds to antigenic determinants on band 3. Unlike RBC autoantibodies from antiglobulin-positive normal blood donors, autoantibodies from patients with autoimmune hemolytic anemia did not show Rh-dependent properties as judged by antigen site density, absence of differential binding to RBC of different Rh phenotypes, failure to immunoprecipitate 30 kd Rh-epitope bearing membrane polypeptides, absence of multiple antibody populations, and the lack of a significant nonadsorbable IgG population that has been associated with the presence of antiidiotypic IgG. The absence of antiidiotypic IgG in hemolytic autoantibodies may be a contributory factor in the development of autoimmune hemolytic anemia.

Aged↗

PubMed1989

Nonhemolytic red cell autoantibodies consist of multiple immunoglobulin G populations directed against complex membrane epitopes.

Immunoglobulin G red cell autoantibodies obtained by elution from six normal blood donors with antiglobulin-positive test results were labeled with iodine 125 and characterized by adsorption studies with primate and human red cells of common and rare Rh phenotype. Adsorbable IgG was determined by exhaustive adsorption with one type of red cell and by cross-adsorption with use of two different types of red cells. All of the labeled autoantibodies studied contained multiple populations directed against epitopes variably expressed on the different types of red cells (both rare and common phenotypes) used for adsorption. At least six antibody populations defining different specificities were identified by cross-adsorption techniques with use of the red cells available. None of the antibody populations was directed against any of the major Rh alloantigens (D, C, E, c, and e) although red cells of the same Rh phenotypes from different donors adsorbed similar quantities of autoantibodies. There was remarkable consistency in the presence of similar antibody populations in all six labeled preparations in spite of the known serologic complexity of red cell autoantibodies. Autoantibody eluates were characterized as either reactive or nonreactive toward Rhnull cells on the basis of their serologic activity. Except for the antibody population specific for Rhnull, all additional specificities identified by adsorption studies were present in both types of eluates. The quantity of each antibody population, however, appeared to be unique for each individual autoantibody donor. A significant fraction (23% to 53%) of the labeled IgG in all of the autoantibodies studied was unabsorbable by any of the red cells used.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibodies↗

PubMed1987

Antiidiotypic IgG crossreactive with Rh alloantibodies in red cell autoimmunity.

IgG autoantibodies eluted from RBCs of antiglobulin positive normal blood donors contained at least two antibody populations, an IgG autoantibody (Ab 1), and an IgG population (Ab 2) that agglutinated RBCs coated with some Rh(D) alloantibodies. Eight of 24 autoantibody eluates tested agglutinated 3 of 10 anti-Rh(D) sensitized RBCs. The agglutinating activity was inhibited specifically by preincubation of the autoantibody eluate with the reactive anti-D. The reaction did not require the Fc domain of the anti-Rh(D), since autoantibody eluates agglutinated RBCs coated with F(ab')2 prepared from the reactive anti-D sera. These findings indicate that the RBCs of some antiglobulin-positive blood donors contain an immunoglobulin auto-antiidiotype (Ab 2) against the RBC autoantibody (Ab 1) which is demonstrable through its cross-reactivity with selected Rh(D) alloantibodies. Identification of auto-antiidiotypes in RBC autoimmunity lends support to the idiotype-antiidiotype network hypothesis of immune regulation and is consistent with the bizarre and complex serology of autoimmune hemolytic anemia. The absence of clinical hemolysis in antiglobulin-positive normal blood donors suggests that immunoglobulin idiotype-antiidiotype interactions may play a role in modulating the effects of RBC autoimmunity.

Antibodies, Anti-Idiotypic↗

PubMed1987

Immunospecific red cell binding of iodine 125-labeled immunoglobulin G erythrocyte autoantibodies.

The primary interaction of autoantibodies with red cells has been studied by using labeled autoantibodies. Immunoglobulin G red cell autoantibodies obtained from IgG antiglobulin-positive normal blood donors were labeled with radioactive iodine and compared with alloanti-D with respect to their properties and binding behavior. Iodine 125-labeled IgG autoantibody migrated as a single homogeneous peak with the same relative mobility as human IgG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric focusing pattern of labeled autoantibodies varied from donor to donor but was similar to that of alloanti-D, consisting of multiple IgG populations with isoelectric points in the neutral to alkaline range. 125I-autoantibody bound to all human red cells of common Rh phenotypes. Evidence for immunospecific antibody binding of the labeled autoantibody was based on variation in equilibrium binding to nonhuman and human red cells of common and rare phenotypes, enhanced binding after red cell protease modification, antiglobulin reactivity of cell-bound IgG comparable to that of cell-bound anti-D, and saturation binding in autoantibody excess. Scatchard analysis of two 125I-autoantibody preparations yielded site numbers of 41,500 and 53,300 with equilibrium constants of 3.7 and 2.1 X 10(8) L X mol-1. Dog, rabbit, rhesus monkey, and baboon red cells were antigen(s) negative by quantitative adsorption studies adsorbing less than 3% of the labeled autoantibody. Reduced ability of rare human D--red blood cells to adsorb the autoantibody and identification of donor autoantibodies that bind to Rh null red blood cells indicated that eluates contained multiple antibody populations of complex specificities in contrast to anti-D, which consists of a monospecific antibody population. Another difference is that less than 70% of the autoantibody IgG was adsorbed by maximum binding red blood cells as compared with greater than 85% for alloanti-D. Evidence obtained indicates that the red blood cell nonadsorbable IgG, some 30%, consists of other IgG populations, one of which represents IgG autoanti-idiotypes to the autoantibody.

Agglutination Tests↗

PubMed1986

Rh antigen immunoreactivity after histidine modification.

125I-anti-D IgG and unlabeled blood group allo-antisera in combination with 125I-protein A were employed in assessing antibody binding to red cells (RBC) treated with histidine reagents. The acylating reagent diethylpyrocarbonate (DEP), the alkylating reagent p-bromophenacyl bromide (pBPB) and the photosensitizer dye Rose Bengal (RB) were used under conditions that usually result in the selective modification of histidine in isolated proteins. Progressive apparent inactivation of the D antigen in ghost membranes occurred with increasing DEP concns, which was not demonstrably reversible by hydroxylamine since this reagent itself inactivated the D antigen. Exposure of red cells to 5 mM p BPB resulted in a 50% decrease in binding of 125I-anti-D IgG. Photo-oxidation of RBC in the presence of Rose Bengal apparently inactivated all the major Rh antigens as detected either by labeled anti-D IgG binding, IgG agglutinating serological reagents, or the binding of 125I-labeled protein A following the sensitization of cells with unlabeled antisera. Under conditions of RB treatment, where hemolysis was absent or minimal, 125I anti-D IgG binding decreased to 38-49% of the level seen in controls. Rose Bengal treatment of R1r RBC revealed varying inactivation of all the Rh antigens, i.e. D 15%, C 89%, c 73%, e 54% inactivated, whereas antibody binding activity of the Fya and Fyb antigens present in the same cell was unaffected. Previous reports as well as the pH profile of anti-D binding have implicated the participation of histidine in Rh antigen expression. Our results are consistent with histidine involvement in Rh activity. Whether Rh antigens have essential histidine(s) involved directly in epitope structure, or instead depend on a critical histidine(s) at the lipid-protein interface that modulates antigen expression remains to be determined.

Diethyl Pyrocarbonate↗

PubMed1982

Nonenzymatic glucosylation of low-density lipoprotein alters its biologic activity.

Human low-density lipoprotein (LDL) was glucosylated by incubation in vitro with glucose (20-80 mM) with or without addition of cyanoborohydride. The incorporation of covalently bound glucose was linear over time, and amino acid analysis showed the presence of glucosyllysine residues. The glucosylated LDL (glc LDL) moved more rapidly than normal LDL on agarose electrophoresis. The rate of degradation of 125I-labeled glucosylated LDL (glc LDL) by cultured human fibroblasts was reduced compared with that of native I-LDL, the difference increasing with extent of glucosylation. Effects were seen with blockage of as few as 6-15% of the LDL lysine residues; high-affinity degradation was completely lost when one-third of the lysine residues were blocked. Conjugation of LDL with glucose-6-phosphate also blocked high-affinity uptake and degradation. Whereas native LDL uptake inhibited the activity of beta-hydroxy-beta-methylglutaryl coenzyme A reductase and stimulated acyl coenzyme A:cholesterol acyltransferase activity, glc LDL had no effects on these enzymes. The fractional catabolic rate of glc LDL in guinea pigs was reduced. Degradation of glc LDL by mouse peritoneal macrophages was not significantly faster than that of native LDL. Finally, the presence of glc LDL in human plasma was demonstrated. Preliminary data show that 1.3% of lysine residues in normal LDL and 2-5.3% of lysines in diabetic LDL were glucosylated. Since, like other plasma proteins, LDL undergoes glucosylation in diabetes, its turnover and sites of catabolism may differ from normal and this may be relevant to the accelerated atherosclerosis of diabetes.

Amino Acids↗

PubMedDate not supplied

Immunoreactivity of the Rho(D) antigen in cytoskeleton-free vesicles.

Skeleton-free microvesicles derived from D-positive red cells immunospecifically bind anti-D. Anti-D saturation binding yielded an equilibrium constant value (K = 1-2 X 10(8) I mole-1) similar to intact red cells. The vesicles contained band 3, the major sialoglycoproteins and, at high protein loads, low-molecular-weight polypeptides. Anti-D binding was referenced to total protein, phospholipid, and band 3 content to determine whether there was nonrandom segregation of the D antigen into vesicles. Selective segregation of the D antigen into vesicles could not be demonstrated unequivocally based on the methods and the assumptions of this study. The results, however, indicate that D reactivity does not require a membrane skeletal association.

Anion Exchange Protein 1, Erythrocyte↗