Ce Antigens Research Articles

RH diversity in Mali: characterization of a new haplotype RHD*DIVa/RHCE*ceTI(D2).

Knowledge of RH variants in African populations is critical to improving transfusion safety in countries with populations of African ancestry and to providing valuable information and direction for future development of transfusion in Africa. The purpose of this report is to describe RH diversity in individuals from Mali. Blood samples collected from 147 individuals self-identified as Dogon and Fulani were analyzed for Rh antigens and alleles. The most common RHD allele variant was RHD*DAU0. Five predicted partial-D phenotypes were attributed to RHD*DAU3 or RHD*DIVa. Neither RHD*DAR nor RHD*DIIIa was found. Investigation of RHCE revealed three predicted partial-e antigens encoded by RHCE*ce(254G) in trans to RHCE*cE. Regarding C antigen, 28 Fulani typed as C+ and 16 of 28 harbored at least one RHCE*Ce-D(4)-ce, two being homozygous and predicted to show a rare RH:32,-46 phenotype. A new RHCE*ceTI with replacement of Exon 2 by RHD (RHCE*ceTI(D2)) was identified in Dogon and was identified by inheritance study to be in cis to RHD*DIVa. These samples typed C- with anti-C polyclonal antibody and monoclonal antibodies (MoAbs) MS24, P3X2551368+MS24, and MS273, but positive with anti-RhCe MoAb-BS58. The same pattern was observed in sample with RHD*DIVa/RHCE*ceTI. Our survey indicated an uneven distribution of RH variant alleles between Dogon and Fulani, suggesting that study in well-documented cohorts is warranted. A high incidence of predicted partial-C phenotype encoded by RHCE*Ce-D(4)-ce was found in Fulani. Further study will also be needed to clarify the clinical significance of the new DIVa/ceTI(D2) haplotype encoding partial D and variant ce antigens.

Alloanti‐c (RH4) revealing that the (C)ces haplotype encodes a partial c antigen

In the Rh blood group system, published observations showed that the c antigen has the fewest variant forms of the principal antigens in this system. The partial nature of the c antigen was only reported in c+ Rh:-26 persons and to be associated with the ce(s)(340) allele. This study reports the first case of alloanti-c related to a (C)ce(s) haplotype. Serologic and genetic studies were performed on blood samples of a multitransfused 40-year-old African patient with sickle cell disease displaying a DCcee phenotype. Red blood cells (RBCs) of the patient displayed normal expression of C, c, e, ce antigens either with routine reagents or with monoclonal antibodies. Analyses of DNA and Rh transcripts showed that the patient carried a (C)ce(s)/DCe genotype. The patient's serum contained anti-D, anti-c, anti-E, anti-e, anti-V, anti-Js(a), and anti-S. Anti-c was isolated from the mixture of antibodies by using absorption and adsorption-elution techniques. Anti-c provided consistent reactions with c+ RBCs. Reactions were stronger with c+ ce+ RBCs than with c+ ce- RBCs. No agglutination of RBCs from individuals carrying a homozygous (C)ce(s) genotype was observed. These data provide the evidence that anti-c in our patient was an alloanti-c and, consequently, that (C)ce(s) haplotype encodes a partial c antigen. The clinical significance of anti-c related to this haplotype should be evaluated in the future.

Molecular basis for the RhD negative phenotype in Chinese

RhD negative individuals are rare and only account for 0.3-0.5% of Taiwanese population. There are some variations among Chinese RhD negative individuals and the molecular basis of these variants is unknown. Two hundred and four RhD negative DNA samples were investigated by a modified polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) and RT-PCR. Several representative cases were further studied using Southern blot analysis. Three types of genetic change in RhD negative individuals were discovered in the Taiwanese population. The most common one, accounting for 150 (73.5%) of 204 cases, was a deletion of the D gene with expression of normal ce or cE antigens (72.5% ce, and 1.0% cE), which were produced by the ce or cE allele of the RHCE gene. The second one, Del, was a deletion of 1013 bp between introns 8 and 9 including exon 9 of the RHD gene. This type accounted for 41 (20.1%) of 204 D negative individuals. The third type was caused by genomic rearrangement around intron 2 and intron 9 between the RHD and RHCE genes and it results in a hybrid gene (D-CE-D) with exons 1, 2 and 10 belonging to the RHD gene. This type of recombination accounted for 13 (6.4%) of 204 D negative individuals. The RhD negative variants found in this study are combination of two of the three alleles, described above. The PCR methods, which detect the differences in introns 1, 2 and 4 or exon 7 for differentiating D positive and D negative, are not reliable methods for studies in the Chinese population.

Reactivity of autoantibodies of autoimmune hemolytic anemia with recombinant rhesus blood group antigens or anion transporter band3.

The specificity of autoantibodies in autoimmune hemolytic anemia (AIHA) has been studied using the serological procedure and immunoprecipitation technique with rare phenotype red cells. We attempted to analyze specificity using recombinant rhesus (Rh) blood group and band3 antigens expressed on erythroleukemic cell lines, KU812E. The autoantibody eluates were isolated by the acid elution procedure from the red cells of 20 AIHA patients. The recombinant Rh antigens, RhD, cE, ce, CE, and chimera antigens CE-D and D-CE, were obtained by retroviral cDNA transduction into KU812E cells, and the cell line expressing the antigens was cloned. Band3 cDNA was also obtained and introduced into KU812E and cloned KU812 expressing RhcE. The reactivities of AIHA eluates with recombinant Rh and band3 antigens were studied by flow cytometry. Fifteen eluates reacted with at least one of the RhcE, ce, or CE antigens, and four eluates reacted with RhD. Seven eluates with strong Rh specificity were studied further using chimera antigen. Five eluates showed reduced or lost reactivity, although two eluates reacted identically with the chimera antigens as wild type. These results indicated that conformational epitopes constituted by RhD or CE specific exofacial peptide loops are important for autoantibodies in most cases. Seven eluates reacted with band3, five exclusively. The coexpression study of RhcE and band3 did not enhance the expression of either antigen nor the reactivity with patient eluates, indicating that association of Rh and band3 was not involved in the appearance of autoantigen.

Expression of C antigen in transduced K562 cells.

The Rh blood group system is involved in HDN and transfusion reactions. A retrovirus-expression system was previously used to show that polypeptides carrying the Rh blood group antigens are encoded by the RHD and RHCE genes. This study investigated the structure of the C antigen. K562 cells were transduced with full-length cDNA encoding Ce and CE antigens, and the expression of C, e, and E antigens was examined by flow cytometry using MoAbs. The importance of Cys16 in C antigen expression was examined by utilizing site-directed mutagenesis to convert Cys16 to Trp in cDNA encoding Ce and CE before expression in K562 cells. When K562 cells were transduced with cDNA that was predicted to encode Ce antigens, clear reactivity with anti-e and anti-C was obtained. In contrast, K562 cells transduced with cDNA that was predicted to encode CE antigens gave strong reactivity with anti-E but failed to react with two examples of anti-C. A third example of anti-C gave weak reactivity. When cDNA encoding Ce antigens was mutated to encode Trp16, one example of anti-C had the same reactivity with the mutated polypeptide as with the wild-type molecule, but reactivity with two other anti-C examples was reduced by 50 percent. The nature of polymorphic residue 226 (proline when E is expressed, alanine when e is expressed) has a marked effect on the epitopes recognized by the three C MoAbs studied. The presence of Cys16 in Ce polypeptides influences the presentation of the C epitope recognized by two of the three MoAbs. These experiments provide the first direct demonstration that C and E/e antigens can be expressed on the same polypeptide.

DAR, a New RhD Variant Involving Exons 4, 5, and 7, Often in Linkage With ceAR, a New Rhce Variant Frequently Found in African Blacks

The highly polymorphic Rh system is encoded by 2 homologous genesRHD and RHCE. Gene rearrangements, deletions, or point mutations may cause partial D and CE antigens. In this study, a newRHD variant, DAR, and a new RHCE variant, ceAR, are described in 4 Dutch African Blacks. Serologically, DAR showed weaker reactions with a monoclonal antibody and polyclonal antiserum against D. The DAR phenotype was characterized by complete loss of at least 9 of 37 Rh D epitopes. Erythrocytes expressing ceAR were all typed as VS−, V+. DNA analysis showed a partial D allele with only 3 mutations: C602G (exon 4), T667G (exon 5), and T1025C (exon 7). The ceAR allele carried G48C (exon 1), a hybrid exon 5 (A712G, C733G, A787G, and T800A), and A916G (exon 6). To study the frequency of these variants, 326 South-African Blacks was screened genomically. Of the 326 donors, 16 (4.9%) carried the DAR allele, 20 (6.1%) the ceAR allele, and 14 (4.3%) both mutated alleles. Five of these donors (1.5%) had the DAR phenotype, indicating that they carried the DAR allele homozygously or next to a D-negative allele. Immunogenicity of the D antigen for individuals with the DAR phenotype was proven, because 1 of the 4 Dutch individuals produced allo-antibodies against D after multiple transfusions with D-positive blood. In a multiethnic society, the prevalence of this D phenotype will increase and is therefore relevant in transfusion practice and in prevention of hemolytic disease of the newborn.

DAR, a New RhD Variant Involving Exons 4, 5, and 7, Often in Linkage With ceAR, a New Rhce Variant Frequently Found in African Blacks

AbstractThe highly polymorphic Rh system is encoded by 2 homologous genesRHD and RHCE. Gene rearrangements, deletions, or point mutations may cause partial D and CE antigens. In this study, a newRHD variant, DAR, and a new RHCE variant, ceAR, are described in 4 Dutch African Blacks. Serologically, DAR showed weaker reactions with a monoclonal antibody and polyclonal antiserum against D. The DAR phenotype was characterized by complete loss of at least 9 of 37 Rh D epitopes. Erythrocytes expressing ceAR were all typed as VS−, V+. DNA analysis showed a partial D allele with only 3 mutations: C602G (exon 4), T667G (exon 5), and T1025C (exon 7). The ceAR allele carried G48C (exon 1), a hybrid exon 5 (A712G, C733G, A787G, and T800A), and A916G (exon 6). To study the frequency of these variants, 326 South-African Blacks was screened genomically. Of the 326 donors, 16 (4.9%) carried the DAR allele, 20 (6.1%) the ceAR allele, and 14 (4.3%) both mutated alleles. Five of these donors (1.5%) had the DAR phenotype, indicating that they carried the DAR allele homozygously or next to a D-negative allele. Immunogenicity of the D antigen for individuals with the DAR phenotype was proven, because 1 of the 4 Dutch individuals produced allo-antibodies against D after multiple transfusions with D-positive blood. In a multiethnic society, the prevalence of this D phenotype will increase and is therefore relevant in transfusion practice and in prevention of hemolytic disease of the newborn.

Caractérisation et validation d'un anticorps monoclonal humain anti-C

Peripheral lymphocytes were obtained from an immunized woman against C and Ce antigens. Punction was realized 14 days after childbirth. After hetero-hybridization, a unique cell line continued secreting a monoclonal IgM antibody. Serological characterization of this antibody was determined by direct agglutination tests against 150 native and enzyme treated red blood cells including some rare phenotypes. This antibody was specific of C determinant of the Rh system. It showed strong reactions by saline and enzymatic technics, against C positive cells from Ce positive and Ce negative patients. The validation was performed by a manual direct agglutination test in saline (tube) and by an automated-hemagglutination test (in microplate) against 2,500 patients samples. No discrepancy was observed. This monoclonal IgM anti-C could be used as a potent reagent and since one year, about 30,000 patients and pregnant women have been phenotyped in our laboratory successfully.

Towards a unified theory for immunogenetic systems. III the serologic uncertainty principle (SUP): A new look at some contemporary cis and trans mysteries

It is unambiguously demonstrated that a large number of “pseudo-problems” or law-like effects are generated when two different antibodies within an immunogenetic system are, erroneously, regarded and labelled as simple or “monospecific (e.g. anti-A and anti-B). Accordingly, they will be believed to have non-overlapping reaction ranges or, in set terms, as being not comparable and disjoint (NCD-relation). As an example, the occurrence of so-called cis and trans genes and antigens in the ABO and Rh systems are briefly discussed in the contemporary (simple-simple and simple-complex) conceptual frameworks (theories and languages). The so-called serologic uncertainty principle (SUP) is applied to these pseudo-problems whereby they become completcly solved (i.e. eliminated). Briefly, SUP is based on the postulate that two “basic” antibodies within a specified “system” should always be regarded as complex or cross-reacting with partially overlapping reaction ranges. Accordingly, two such basic antibodies (e.g labelled anti-AX and anti-BX) are, in set terms, not comparable and not disjoint (NCND-relation). In such a complex-simple SUP framework (theory and language), the currently “mysterious” cis AB blood group in the ABO system (or the “improperly behaving” cis CE; Ce; cE; ce antigens in Rh) simply becomes a mono-specific, mono-factorial and mono-symbol main antigen (X) which happens to “cross-react” with two NCND-related antibodies (e.g. anti-A(X) and anti-B(X); anti-C(X) and anti-E(X)). Accordingly, the previous ad hoc assumptions of “jumping genes (e.g. crossing-over), strange cis/trans “laws” and atypical serologic behavior of cis antigens (relative to their trans “partners”) are not required in SUP. The anatomy of the radical conceptual framework shift(s) accomplished through the transition from the contemporary dogma of simple and hence NCD-related antibodies to the new SUP doctrine of complex and NCND-related antibodies is analyzed in some detail.

日本腦炎ワクチンの研究に関連して調べられたマウス腦と鷄胎兒との比較

Between mouse brain (MB) and 12-day-old chick embryo body (CE) tissue, as materials for the preparation of JAPANESE ENCEPHALITIS vaccine, several differences were found:1) Total Nitrogen content of MB tissue was 2.3 times while the supernate of MB centrifuged at 2500r.p.m. for 15 minutes contained 1.2 times as much Nitrogen, 2) total solids of MB was 2.6 times, 3) mouse intracerebral titers of infected MB tissue were from 6.3 to 200 times, 4) and calculated mouse i. c. LD 50 per mg Nitrogen of MB supernate was from 5, 4 to 170 times, all the above mentioned, approximately, as compared with those of CE tissue. 5) In the cross complement fixation tests using MB and CE antigens with the corresponding immune guinea pig sera, the reaction in homologous relation were stronger than in heterologous relation. 6) Any significant differences in neutralization indices could not be found in cross neutralization test using the same immune guinea pig sera as in c. f. test, and the corresponding both viruses. 7) In cross immunization tests in mice which were vaccinated with MB and CE vaccine and challenged with both viruses, the protection in homologous relation was not necessarily stronger than that in heterologous relation. 8) The protective power of CE vaccine was disproportionnally weaker than that of MB vaccine on the basis of infective titer before inactivation of vaccine. 9) The heated infected mouse brain suspension have a tendency of lowering LD50 titer when added to MB or CE virus in infectivity titration. The same was not observed in the heated CE suspension. This inhibiting effect on virus seems to be partly nonspecific.