Immunochemistry of Ii-active glycosphingolipids of erythrocytes.

Fractions of complex glycosphingolipids were prepared from adult, cord, and i phenotype erythrocytes by the method elaborated for the isolation of poly(glycosyl)ceramides. In contrast to poly(glycosyl)ceramides which comprise on the average 30 glycosyl units and about 5 branching points, i.e. 3,6-di-O-substituted galactopyranosyl residues, per mole of glucose, complex glycosphingolipids from cord and i erythrocytes comprise 6 and 15 glycosyl units respectively and only 0.7 branching points. The latter substances exhibited also a high i activity which was not detected in poly(glycosyl)ceramides. Erythrocyte membranes were labeled with radioactive N-acetylgalactosamine (GalNAc) from UDP-GalNAc using a purified A-blood-group gene-specified transfered of GalNAc. It was found that electrophoretic mobilities in dodecylsulfate-gel electrophoresis of all glycoconjugates which accepted GalNAc were increased in i as compared to I membranes. We conclude that the absence of highly branched glycosphingolipids in cord and i erythrocytes as well as the reduction of apparent molecular weights of the glycoconjugates, which are substrates for A-gene-specified transferase of GalNAc, result from a single cause, that is an inadequacy of the biosynthetic process which is responsible for the formation of GlcNAc1 leads to 6Gal structures.

Subtypes of i demonstrated by the use of atypical Ii cell types and inhibition studies.

Haemagglutination, absorption and elution studies with several anti-i sera and atypical Ii red cell types and Oh cells were carried out and three subtypes of specificity were demonstrated. The fourth i subtype was demonstrated by an inhibition method using human serum and amniotic fluid as a source of i substance.

Immunoglobulins in maternal serum and in amniotic fluid in pregnancy complicated by rhesus alloimmunization.

The concentration of IgG, IgA, and IgM immunoglobulins was checked in sera of Rh-immunized and nonimmunized pregnant women. The amniotic fluid of immunized women was also investigated. The significant decrease of IgG, accompanied by significant increase of IgA immunoglobulin was revealed in the sera of immunized patients. In non-concentrated amniotic fluid only IgG and IgA immunoglobulins were found.

The evaluation of extended analysis of serum, amniotic fluid and cardiotocography in the management of pregnancy complicated by immunization in Rh or Kell antigens.

The estimation of protein and bilirubin concentration together with checking the activity of LDH, AspAT, AlAT and Ech in maternal serum and amniotic fluid was performed with intention to improve the accuracy of antenatal prediction in hemolytic disease of newborns. With the same intention, the cardiotocography was introduced. After study on forty-six pregnancies, the usefulness of estimation of optical density, protein and bilirubin concentration in amniotic fluid and calculation of bilirubin/protein ratio was confirmed. The other investigated methods appeared useless.

The use of plasmapheresis in pregnancy with antenatal prediction of a serious hemolytic disease due to Rh immunization.

Plasmapheresis was performed on twelve women with antenatal prediction of severe hemolytic disease due to Rh or Kell alloimmunization. No complications in mothers or newborn infants due to this treatment were observed neither during pregnancy nor in post-deliver period. In two patients the increase of antibody titer was not prevented. In four cases the fetus was not saved.

Isolation and partial characterization of I antigen from I adult erythrocyte membranes.

The i-active substance was isolated from i adult and I int erythrocyte membranes by butanol-water extraction and was purified by SDS-gel filtration and thin layer chromatography of peracetylated samples. The i-active substances from both types of erythrocytes showed also IF activity. In antigen isolated from i adult red cells the IF activity was lower than i activity. On the other hand, the antigen of I int red cells showed higher IF than i activity. The chemical characterization of isolated antigens indicated their glycosphingolipid character.

Further investigation of Ii phenotypic variants.

I and i subtypes were investigated in erythrocytes with depressed expression of I antigen. It was confirmed that both, I and i antigens are heterogeneous. The depressed i status was found in all abnormal I phenotypes, but no direct correlation between i status and I subtype was found.

Activity of B-gene-specified galactosyltransferase in individuals with Bm phenotypes.

Blood group B-gene-specified alpha-galactosyltransferase was determined in serums and erythrocyte membranes of several related persons with Bm phenotypes. The level of this enzyme in serums was similar to, but in erythrocyte membranes much lower than that found in normal B and AB persons.

Reactions of erythrocyte glycoproteins and their degradation products with various anti-I sera.

Three fractions of erythrocyte glycoproteins obtained from Sepharose 4-B chromatography were tested for I activity with ten serologically differentiated anti-I sera. The most active was fraction I, eluted at the void volume and containing the lowest amount of alkali-labile oligosaccharide chains. The desialization of glycoproteins increased their activity toward anti-I-s and anti-I-D sera, and did not change or decreased the activity toward anti-I-F sera. The most abundant fraction II (major sialoglycoprotein of erythrocyte membranes) showed no or only a very weak I activity, but I-active glycopeptides were isolated from products of digestion of fraction II with trypsin. The major product of digestion, sialoglycopeptide IIT-2 showed I activity only after alkaline elimination of alkali-labile oligosaccharide chains. The results indicate that I receptors are present in hindered form on apparently I-inactive components of erythrocyte membrane.

Serological classification of anti-I sera.

Twelve anti-I sera were examined by means of broad panel of cells: OI, BI, OhI, Oicord, Oiadult, BI-F. Titration, fixation-elution, and haemagglutination-inhibition tests, and comparison of agglutination of untreated and neuraminidase-treated erythrocytes allowed to establish the specificity of the sera. For some sera the serological heterogeneity was revealed, which consisted in the following combinations of two or more kinds of specificity: anti-I-D plus anti-I-F, anti-I-D plus anti-i, anti-I-D plus anti-I-S, anti-I-D plus anti-I-F plus anti-I-S. Methodical problems of classification of anti-I sera are discussed.

HL-A matching in thrombocyte and leukocyte transfusions.

A panel of 150 Rh+ and Rh-- donors was tested by the NIH-microcytotoxicity test against 90 antisera determing 21 HL-A antigens. For platelet and leukocyte transfusions, donors compatible in ABO and Rh groups as well as in HL-A antigens, were used. In some cases the donor and recipient were compatible in three HL-A antigens, in other cases the degree of compatibility was less satisfactory. Platelet and leukocyte concentrates were prepared by double thrombo- and leukocyte-pheresis. Before transfusions the cross cytotoxicity test with donor's lymphocytes was performed. No transfusion accidents occurred; in some patients a beneficial effect was noticed.