Rhesus System Research Articles

Effect of Producer Cell Line on Functional Activity of Anti-D Monoclonal Antibodies Destined for Prevention of Rhesus Sensitization

The ability of anti-D antibodies to cause antigen-specific immunosuppression depends on their interaction with low-affinity Fcgamma-receptors. Human monoclonal antibodies to D antigen of the rhesus system were investigated by antibody-dependent cytotoxicity assay in order to estimate their ability to induce hemolysis mediated by low-affinity Fcgamma receptors. We demonstrate that affinity of monoclonal antibodies to receptors of this type does not depend on primary structure of Fc-fragment, but depends on the producer cell line which expresses the antibodies. Monoclonal IgG1 antibodies interacting with FcgammaRIIa and FcgammaRIII lost this property, if they were secreted by human-mouse heterohybridoma, but not by human B-cell line. On the opposite, monoclonal antibodies that could not activate low-affinity Fcgamma receptors were highly active after human cells fusion with rat myeloma YB2/0. Hemolytic activity of IgG3 remained unchanged after fusion of human cells with rodent cells.

Immunological biochips for studies of human erythrocytes

Immunological microarrays (biochips) for detecting erythrocyte surface antigens, viz., blood group antigens (A, B, 0) and Rhesus system antigens (D, E, e, C, and c), are described. The biochips represent trans- parent plastic supports onto which 1.5-mm spots of specific immobilized antibodies (IgM) are coated in differ- ent dilutions. The volume of tested blood samples is rather small (1-2 µ l). Binding of erythrocytes to antibodies immobilized on the biochips is specific and allows further morphological analysis of bound cells. Analysis of the dynamics of cell detachment from biochip spots using a microfluidic chamber at different flow rates of the washing solution showed that combination of a biochip with a microfluidic chamber is a promising approach to concentration of cells of various immunotypes even if their content in the mixture is very low.

Fetal microchimerism in Hashimoto’s thyroiditis: a quantitative approach

Fetal microchimerism (MCH) has been implicated in the etiology of autoimmune diseases such as autoimmune thyroiditis. The goal of the study was to reliably estimate the number of fetal engrafted cells and to further investigate factors influencing the development of MCH. Quantitative real-time PCR amplification using Y-chromosome specific (DYS14) and autosomal (beta-globin) loci was performed on thyroid gland specimens. Furthermore, we compared the distribution of ABO and rhesus systems in mothers with and without blood MCH in relation to the blood groups of the children. MCH was detected in eight of 21 Hashimoto patients in a frequency range of 15 to 4900 male cells per 100,000 total cells (median 97 cells), but in none of 17 healthy thyroid glands. In a third group, consisting of 18 nodular goiters, only one sample was positive (182 male cells/100,000 total cells). No woman who had not had a prior pregnancy with a male fetus showed MCH. Mothers both with and without MCH showed the same rate of mother/child incompatibilities for the ABO and rhesus systems. The percentage of microchimeric cells varies to a great extent in Hashimoto's thyroiditis, and this phenomenon can occur in nodular goiter in rare instances, but it appears to be absent from normal thyroid glands. Nevertheless, the biological significance of MCH remains unclear. Moreover, we have concluded that the tested blood group systems (as opposed to their role in graft vs host disease after transplantations) have no effect on fetal MCH.

Red blood cell alloantibodies after transfusion: factors influencing incidence and specificity

Alloimmunization after exposure to red cell (RBC) alloantigens depends on genetic and acquired patient-related factors, dose and route of administration, and the immunogenicity of the antigen, but exact kinetics are still unknown. A 5-year retrospective multicenter study analyzing factors influencing the rate and specificity of RBC alloimmunization was performed, with special emphasis on the time interval between transfusion event and antibody detection. Included were clinically significant alloantibodies against the Rhesus, Kell, Duffy, Kidd, and MSs blood group systems. Multivariate analysis involving 1710 immunized patients revealed that time interval between transfusion and antibody tests was strongly associated with the antibody specificity. Anti-Jk(a) and anti-Jk(b) were predominantly found in patients tested within 3 months, whereas anti-K and anti-Fy(a) were the most encountered antibodies at more than 5 years after transfusion. Of all immunized patients, new antibodies were detected within 14 days after transfusion in 299 patients (16.8%) and in 1479 patients (83.2%) after more than 14 days. Fifty percent of transfusion recipients were retested for alloimmunization because of a new transfusion indication. Eleven of 2932 patients (0.4%) retested up to 3 days after transfusion had formed new antibodies. The time interval between transfusion and antibody test was associated with RBC antibody specificity. Because RBC antibody tests after transfusion are not routinely performed, many antibodies may (not) be detected at the time of a new transfusion event, posing the transfusion recipient at risk for transfusion delay or a (delayed) hemolytic transfusion reaction. Routine RBC antibody screening at set time intervals after transfusion would reduce these risks.

FREQUENCY OF VARIOUS RH ANTIGENS IN BASRAH PROVINCE, IRAQ

Knowledge of the frequencies and the phenotype of Rhesus system is very important for Blood Bank and transfusion service policies. This study was conducted on 2875 blood samples from Blood Bank in Teaching Hospital in Basrah to study different Rh phenotype in blood donors. The result showed that the frequency of Rh positive was found to be (93.1%) and Rh negative was (6.99%). The Rh phenotypes in 87 Rh negative blood samples present in decreasing order of frequency were as follows: rr, r1r, r11r, r1r11, r1r1, r11r11, ry . The results of this study were compared with the results of other studies.

Risk factors for alloimmunization by patients with sickle cell disease

Blood transfusion in patients with sickle cell disease (SCD) is limited by the development of alloantibodies to erythrocytes. In the present study, the frequency and risk factors for alloimmunization were determined. Transfusion records and medical charts of 828 SCD patients who had been transfused and followed at the Belo Horizonte Blood Center, Belo Horizonte, MG, Brazil, were retrospectively reviewed. Alloimmunization frequency was 9.9% (95% CI: 7.9 to 11.9%) and 125 alloantibodies were detected, 79% of which belonged to the Rhesus and Kell systems. Female patients developed alloimmunization more frequently (P = 0.03). The median age of the alloimmunized group was 23.3 years, compared to 14.6 years for the non-alloimmunized group (P < 0.0001). Multivariate analyses were applied to the data for 608 hemoglobin (Hb) SS or SC patients whose number of transfusions was recorded accurately. Number of transfusions (P = 0.00006), older age (P = 0.056) and Hb SC (P = 0.02) showed independent statistical associations with alloimmunization. Hb SC patients older than 14 years faced a 2.8-fold higher (95% CI: 1.3 to 6.0) risk of alloimmunization than Hb SS patients. Female Hb SC patients had the highest risk of developing alloantibodies. In patients younger than 14 years, only the number of transfusions was significant. We conclude that an increased risk of alloimmunization was associated with older patients with Hb SC, specially females, even after adjustments were made for the number of transfusions received, the most significant variable.

Alloimmunization to red blood cell antigens after universal leucodepletion. A regional multicentre retrospective study

Leucodepletion has been shown to reduce human leucocyte antigen immunization, but studies on the effect of leucodepletion on red cell alloimmunization reported discordant results. We conducted a retrospective multicentre study to determine whether prestorage filter leucodepletion alters the development of clinically significant red blood cell alloimmunization against the Rhesus, Kell, Duffy, Kidd and MSs blood group systems. Two periods were investigated, 2 years before and 2 years after universal leucodepletion. Comparisons were made between the transfused patient cohorts. To control for changes not related to leucoreduction, we compared antibody incidence with antibody prevalence in the two study periods. Newly detected antibodies (n = 4770) were found in 4115 patients from 19 participating hospitals. Of these, 857 antibodies in 659 patients were because of transfusions given in the study periods. The immunization risk was 0.13% for both periods. No differences were found regarding incidence of new antibodies, nor for patients regarding age, sex, previous antibodies, multiple antibodies, additional antibodies, number of transfusions, transfusions episodes and days from transfusion to date of immunization. In conclusion, compared with buffy-coat leucoreduction, universal prestorage filter leucodepletion did not alter the development of clinically significant red blood cell alloimmunization.

Serological researches in the south of Moldavia in connection with the problem of the ethnogeny of the Gagauzes, the Moldavians and the Bulgarians

With the aid of data of frequencies of genetically determined blood group systems, the authors have tried to show the basic ethno-genetic directional patterns in Southern Moldavia and in the Dniester-Carpathian-Danubian region in its entirety. Blood Groups A1A27B0, RHESUS, MN, and KELL have been determined in six random samples from four Gagauz villages (n = 330), one Moldavian village (n = 101) and one Bulgarian village (n = 96). The analysis of gene frequencies demonstrates genetic homogeneity of the total Gagauz population. Statistically reliable differentiation is observed only for the RHESUS system. It is possible now to suppose that the haemotological types of modern Gagauz and Bulgarian populations have been developed on the basis of the Balcanic serogenetic types, partially transformed under influence of gene flow from probably Central Asian or other eastern centers. The position of the Moldavians on the serogenetic map of Europe is less certain, due to a considerable ambiguity in the allele frequencies of the various blood group systems. Founder effects may account for these observations. However, the genetic distance and cluster analyses carried out on the frequencies of the surveyed blood group systems have shown the affinity of Moldavians with the Romanians and Eastern European populations.

Detection of Red Blood Cell Alloantibodies by Gel Centrifugation Test

Background: Gel centrifugation techniques and solid-phase antiglobulin tests have increased sensitivity and specificity for red blood cell antibody (rbc-ab) detection compared to classical tube techniques. In this report we present results of rbc-ab screening and differentiation of patient and donor sera using two variants of gel centrifugation test. Material and Methods: 95,138 sera of 76,693 patients and blood donors of Rostock University Medical School (samples of a 7.5-year period) were screened for rbc-abs by gel centrifugation test (DiaMed ID microtyping system, papain 37 °C, and indirect antiglobulin test with LISS). Positive sera were further differentiated in the same test system. Results: The incidence of alloimmunization was 2.68%. Differentiation of 6,407 positively screened sera resulted in 3,160 unspecific reactions preferably in enzyme test, 532 negative sera, 484 sera containing anti-D following Rhesus prophylaxis, 73 rbc autoantibodies, 103 cases of dubious antibody specificity, and 2,055 cases of rbc alloantibodies with defined single or multiple specificity. Anti-E was most frequently found (27.3%) followed by anti-D (18.1%), anti-Le(a) (12.5%), anti-Cw (9.7%), anti-C (8.7%), anti-K (7.9%), anti-c (3.9%), anti-Fy(a) (2.8%), anti-Jk(a) (1.6%), and further specificities in lower frequency. Especially for detection of antibodies directed against antigens of Rhesus system, the enzyme test was more sensitive than the antiglobulin test. Conclusion: Data of this investigation indicate that differentiation of rbc-abs by enzyme test plays an important role for early detection of Rhesus antibodies. Unfortunately, the enzyme test yields more unspecific reactions than the antiglobulin test. Therefore, unclear test results obtained with enzyme-treated rbcs in gel centrifugation test require critical interpretation.

The use of the expectation-maximization (EM) algorithm for maximum likelihood estimation of gametic frequencies of multilocus polymorphic codominant systems based on sampled population data

Estimation of gametic frequencies in multilocus polymorphic systems based on the numerical distribution of multilocus genotypes in a population sample ("analysis without pedigrees") is difficult because some gametes are not recognized in the data obtained. Even in the case of codominant systems, where all alleles can be recognized by genotypes, so that direct estimation of the frequencies of genes (alleles) is possible ("complete data"), estimation of the frequencies of multilocus gametes based on the data on multilocus genotypes is sometimes impossible, whether population data or even family data are used for studying genotypic segregation or analysis of linkage ("incomplete data"). Such "incomplete data" are analyzed based on the corresponding genetic models using the expectation-maximization (EM) algorithm. In this study, the EM algorithm based on the random-marriage model for a nonsubdivided population was used to estimate gametic frequencies. The EM algorithm used in the study does not set any limitations on the number of loci and the number of alleles of each locus. Locus and alleles are identified by numeration making possible to arrange loops. In each combination of alleles for a given combination of m out of L loci (L is the total number of loci studied), all alleles are assigned value 1, and the remaining alleles are assigned value 0. The sum of zeros and unities for each gamete is its gametic value (h), and the sum of the gametic values of the gametes that form a given genotype is the genotypic value (g) of this genotype. Then, gametes with the same h are united into a single class, which reduces the number of the estimated parameters. In a general case of m loci, this procedure yields m + 1 classes of gametes and 2m + 1 classes of genotypes with genotypic values g = 0, 1, 2, ..., 2m. The unknown frequencies of the m + 1 classes of gametes can be represented as functions of the gametic frequencies whose maximum likelihood estimations (MLEs) have been obtained in all previous EM procedures and the only unknown frequency (Pm(m)) that is to be estimated in the given EM procedure. At the expectation step, the expected frequencies (Fm(g) of the genotypes with genotypic values g are expressed in terms of the products of the frequencies of m + 1 classes of gametes. The data on genotypes are the numbers (ng) of individuals with genotypic values g = 0, 1, 2, 3, ..., 2m. The maximization step is the maximization of the logarithm of the likelihood function (LLF) for ng values. Thus, the EM algorithm is reduced, in each case, to solution of only one equation with one unknown parameter with the use of the ng values, i.e., the numbers of individuals after the corresponding regrouping of the data on the individuals' genotypes. Treatment of the data obtained by Kurbatova on the MNSs and Rhesus systems with alleles C, Cw, c, D, d, E, e with the use of Weir's EM algorithm and the EM algorithm suggested in this study yielded similar results. However, the MLEs of the parameters obtained with the use of either algorithm often converged to a wrong solution: the sum of the frequencies of all gametes (4 and 12 gametes for MNSs and Rhesus, respectively) was not equal to 1.0 even if the global maximum of LLF was reached for each of them (as it was for MNSs with the use of Weir's EM algorithm), with each parameter falling within admissible limits (e.g., [0, min(PN,Ps)] for PNs). The chi 2 function is suggested to be used as a goodness-of-fit function for the distribution of genotypes in a sample in order to select acceptable solutions. However, the minimum of this function only guarantee the acceptability of solutions if all limitations on the parameters are met: the sum of estimations of gametic frequencies is 1.0, each frequency falls within the admissible limits, and the "gametic algebra" is complied with (none of the frequencies is negative).

Molecular cloning and expression of rhesus macaque and sooty mangabey interleukin 16: biologic activity and effect on simian immunodeficiency virus infection and/or replication.

Interleukin 16 (IL-16) has been shown to diminish HIV and SIV replication through inhibition of HIV and SIV mRNA transcription. To evaluate its role further, we compared IL-16 cloned from disease-susceptible rhesus macaques and disease-resistant sooty mangabeys. Recombinant rhesus macaque (rr) IL-16 was compared with recombinant sooty mangabey (rm), human, and other nonhuman primate IL-16 sequences and evaluated for its ability to induce chemotaxis and inhibit the mixed lymphocyte response (MLR). Also, rrIL-16 and rmIL-16 were evaluated for suppression of SIVmac251, which replicates efficiently in T cells and monocyte/macrophages (dual tropic), and cloned SIVmac239, which replicates efficiently in T cells (T tropic). Sequence comparison of rrIL-16 and rmIL-16 with human IL-16 showed >97% amino acid identity. Biocharacterization of rrIL-16 revealed potent induction of chemotaxis (p < 0.05) and marked inhibition of MLR (73 +/- 0.6%,p < 0.05) in rhesus and human cell systems. Using rrIL-16 and rmIL-16, p27 antigen production from PBMCs infected with SIVmac251 was decreased up to 70% (p < 0.05 and p < 0.01, respectively). In similar cultures infected with SIVmac239, rrIL-16 and rmIL-16 reduced p27 levels by 96 and 100%, respectively. These data demonstrate the biologic and antiviral functionality of rrIL-16 and rmIL-16.

Origin of Metazoa: sponges as living fossils.

The phylogenetic position of the phylum Porifera (sponges) is at the base of the kingdom Metazoa. During the past few years not only rDNA sequences but--and this was a major advance--even cDNAs/genes have been isolated and characterized from sponges, especially from the marine demosponge Geodia cydonium, which code for proteins. The analyses of their deduced amino acid sequences allowed a molecular biological approach to solve the problem of monophyly of Metazoa. Molecules of the extracellular matrix/basal lamina, with the integrin receptor, fibronectin, and galectin as prominent examples, cell-surface receptors (tyrosine kinase receptor), elements of sensory systems (crystallin, metabotropic glutamate receptor), and homologs/modules of an immune system (immunoglobulin like molecules, scavenger receptor cysteine-rich, and short consensus repeats, rhesus system) classify the Porifera as true Metazoa. As living fossils, provided with simple, primordial molecules allowing cell-cell and cell-matrix adhesion as well as processes of signal transduction as known in a more complex manner from higher Metazoa, they also show peculiarities not known in other metazoan phyla. Tissues of sponges are rich in telomerase activity, suggesting a high plasticity in the determination of cell lineages. It is concluded that molecular biological studies with sponges as model will not only help to understand the evolution of Protoctista to Metazoa but also the complex, hierarchial regulatory network of cells in higher Metazoa.

Red blood cell alloimmunization in sickle cell disease: the influence of racial and antigenic pattern differences between donors and recipients in Brazil.

Red blood cell (RBC) transfusions are widely used in the management of patients with sickle cell disease (SCD). However, repeated RBC transfusions are often complicated by RBC alloimmunization. To investigate whether the frequency of RBC alloimmunization could be accounted for by racial and RBC phenotype differences between donors and recipients in Brazil, in this study we compared the RBC phenotype of 100 SCD patients with that observed in 120 randomly selected blood donors. A comparison of the RBC phenotype between the two groups revealed a statistically significant increase in the frequency of the C antigen in the donor population (P < 0.01), but no significant difference was observed for the A,B,D,c,E,e,K,k,Fya,M,N,S,s, and Jka antigens. Using standard techniques (indirect antiglobulin test, enzyme treatment, and low-ionic-strength solution) we observed an RBC alloimmunization rate of 12.9% (11/85) in the SCD patients. Fifteen alloantibodies were detected in 11 patients, and most (80%) involved antigens in the Rhesus and Kell systems. This observed RBC alloimmunization rate in SCD patients in Brazil is lower than that reported by studies from North America, suggesting that the requirement for extended antigen-matched RBC transfusion for SCD patients in the setting of a RBC phenotype concordant donor-recipient population may not be cost-effective in some countries.

The R0Har RH:33 phenotype results from substitution of exon 5 of the RHCE gene by the corresponding exon of the RHD gene.

The highly polymorphic Rh (Rhesus) system is encoded by two homologous genes, one encoding the D polypeptide and the other the CcEe polypeptides. Partial D antigens may be caused by gene rearrangements, deletions or point mutations. In this study the molecular basis of R0Har RH:33, a Rh phenotype of low frequency, is described. The R0Har RH:33 phenotype is characterized by partial expression of D, altered expression of e, absence of G and the presence of two antigens of low frequency: Rh33 and FPTT. Southern blot analysis, RHD typing by PCR and sequence analysis of Rh transcripts revealed that the RHD gene is absent in subjects with this phenotype. Apart from the expected RHCE transcripts, a new Rh transcript, RHc(D)(e), was identified in three unrelated individuals expressing R0Har Rh:33. The RHc(D)(e) transcript showed the same sequence as the RHce transcript, with the exception of exon 5, which was substituted by the corresponding exon of the RHD gene. A method for PCR-based genotyping was developed to determine specifically the c(D)(e) haplotype. The c(D)(e) PCR proved to be a reliable alternative method for R0Har RH:33 typing.

CBV during exchange transfusion in infants with erythroblastosis.

Since the introduction of postpartal and prepartal Anti-D-prophylaxis (Bowman et al., 1978; Thornton et al., 1989), the rate of sensibilisation to the rhesus system has decreased in Germany from 1:20.000 before prophylaxis to 1:1.000 today (Maas et al., 1991). There is, however, little chance that the future could hold an even lower incidence of infants with isoimmunisation caused by Rhesus incompatibility, since prophylaxis has a constant failure rate. In addition, there are other types of isoimmunisations caused by other blood groups, especially anti-c, Kell and Duffy, where no prophylaxis is yet possible. Therefore exchange transfusion (ET) will continue to be a part of the postnatal management in neonates suffering from Morbus haemolyticus.

The prevalence of red cell antibodies in pregnancy correlated to the outcome of the newborn: a 12 year study in central Sweden.

All maternal red cell antibodies found during pregnancy in a 12 year period have been compiled. The efficacy of the current antenatal screening and management programme has been ascertained by reviewing the outcome of all newborns to these immunized mothers. Patient selection was carried out by computerised searching for all known records of registered antibodies during the study period. Each mother's obstetric record and her baby's hospital file was studied and relevant clinical treatment and laboratory data on both mother and child was recorded and analysed. Eight hundred and twenty-one alloantibodies were detected in 629 immunized pregnant women with 753 fetuses. An overall antibody incidence of 0.57% was observed which included 373 clinically significant antibodies found in 261 mothers (0.24%). Multiple antibodies were present in 8.2% of all samples. Anti-D, by itself or in combination with other Rh-antibodies, caused more severe forms of hemolytic disease of the newborn (HDN) with 46% of all Rh-positive babies having phototherapy and 29% having exchange transfusion. Three of 18 Fya-positive infants required phototherapy and one required exchange transfusion and in the 16 Kell-positive babies, three required phototherapy and one required exchange transfusions. Few antibodies to blood group antigens other than those in the Rhesus system were found to cause severe HDN. Antibodies that are generally considered non-significant did not cause HDN in this study. All antibodies that induced HDN were detected in time so that adequate measures could be taken to reduce the effects in the newborn. The antenatal screening and management programme currently in use is considered to be reliable.

Blood group antigen survival on freeze-dried erythrocytes and ghosts after isolation.

To screen and identify irregular antibodies, whatever the technique used, fresh erythrocytes (RBCs) are needed to set up the panel. Solid-phase tests using dried blood cells are available, but the technique is based on the adherence of sensitized RBCs, which have a short life span. We have checked antigen survival on membranes with a saline test and an antiglobulin test for two methods to preserve the antigen substrate: freeze-drying of RBCs and preparation of RBC membranes. The different antigens of the ABO, Rhesus, Kell, P, Lewis, MNSs, Lutheran, Duffy, Kidd and Li systems are well recognized on the membranes after isolation and on freeze-dried cells. Demonstration of antigen survival leads us to consider using membranes or freeze-dried cells in new immunological tests.

Molecular genetics of chimpanzee Rh-related genes: Their relationship with the R-C-E-F blood group system, the chimpanzee counterpart of the human rhesus system

As the chimpanzee R-C-E-F blood group system appears to be the chimpanzee counterpart of the human Rhesus (RH) system, we have tried to determine whether chimpanzee Rh-like genes encode R-C-E-F-related proteins. Chimpanzee genomic DNA, digested by any of eight endonucleases and hybridized with three Rh exon-specific probes, exhibits a high degree of polymorphism. Analysis of DNA from unrelated individuals of different R-C-E-F types revealed that the presence of some restriction fragments is correlated with particular R-C-E-F types. The cosegregation of these fragments with R-C-E-F haplotypes was confirmed by family studies. Oligonucleotides complementary to regions flanking human exons were used as PCR primers on chimpanzee DNA; the resulting amplified fragments were identical in size to their human counterparts. Moreover, the nucleotide sequences of the fragments present a high degree of similarity to the corresponding human regions.

Rhesus system polymorphism in the Tunisian population

A survey carried out on 4129 Tunisian blood donors permitted to evaluate the distribution of Rhesus blood group in Tunisia. The haplotypes frequency were as follow, R1: 0,367 r: 0,284 R0: 0,2082 R2: 0,1218 R': 0,00188 r'': 0,0001.

Molecular cloning and primary structure of the human blood group RhD polypeptide.

The RH (rhesus) blood group locus from RhD-positive donors is composed of two homologous structural genes, one of which encodes the Cc and Ee polypeptides, whereas the other, which is missing in the RhD-negative condition, encodes the D protein that carries the major antigen of the RH system. Recently, different splicing isoforms transcribed from the CcEe gene were isolated. We report now the characterization of two other Rh clones, RhII and RhXIII, generated by alternative choices for poly(A) addition sites that were identified as the RhD gene transcripts. That these cDNAs represented the RhD messenger and that the previously described Rh clones were derived from the CcEe gene was demonstrated by amplification of RhII/XIII sequences only from D-positive genomes and by cloning and sequencing of D- and CcEe-specific gene fragments. The predicted translation product of the RhD mRNA is a 417-amino acid protein (M(r) = 45,500) that exhibited a similar membrane organization with 13 bilayer-spanning domains compared with the polypeptide encoded by the CcEe gene. The D and Cc/Ee polypeptides differ by 36 amino acid substitutions (8.4% divergence), but the NH2- and COOH-terminal regions of the two proteins are well conserved. Similarly, five of the six cysteine residues of the Cc/Ee proteins were conserved in the D protein, including the unique exofacial cysteine, which is critical for antigenic reactivity. The sequence homology between the Cc/Ee and D proteins supports the concept that the genes encoding these polypeptides have evolved by duplication of a common ancestor gene.