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C M Westhoff

Publications and source records attributed to C M Westhoff.

At least 19 recordsLinked to original sources
PubMed2006

Transport characteristics of mammalian Rh and Rh glycoproteins expressed in heterologous systems.

The development and use of heterologous expression systems is critical for deciphering the function of mammalian Rh and Rh-glycoproteins. The studies here use Xenopus oocytes, well known for their ability to readily traffic and express difficult membrane proteins, and S. cerevisiae wild-type strains and mutants that are defective in ammonium transport. Data obtained in both of these expression systems revealed that mammalian Rh-glycoprotein-mediated transport (RhAG, RhBG, and RhCG) is an electroneutral process that is driven by the NH4+ concentration and the transmembrane H+ gradient, effectively exchanging NH4+ for H+ in a process that results in transport of net NH3. Homology modeling and functional studies suggest that the more recently evolved erythrocyte blood group proteins, RhCE and RhD, may not function directly in ammonia transport and may be evolving a new function in the RBC membrane. The relationship of Rh and Rh-glycoproteins to the Amt/Mep ammonium transporters is substantiated with functional transport data and structural modeling.

Animals↗
PubMed2006

Serologic and molecular genetic management of a pregnancy complicated by anti-Rh18.

Antibodies, such as anti-Rh18 (Hr/Hr(S)), that react with the common products of RHCE can cause HDN as well as severe hemolytic transfusion reactions. Individuals with anti-Rh18 antibodies can have different RHCE genetic backgrounds; therefore, sera and RBCs from these individuals may cross-react. In these situations, genotyping may be the best method to determine compatibility. We report a 26-year-old pregnant Puerto Rican woman who presented at 31 weeks' gestation with anti-E and anti-Rh18 in her serum. No potential donors were identified among family members or within the American Rare Donor Program; therefore, a unit of the patient's RBCs was collected one week before her planned caesarian section. To improve our ability to supply blood for this patient in the future, molecular testing was performed. The patient was found to be homozygous for an RH haplotype in which a variant RHD*DAR, is linked to a variant RHCE*ceAR. The DAR-ceAR haplotype has been described in Dutch-African populations, but this is the first report of an individual self-identified of Hispanic ethnicity. This case report demonstrates the clinical importance of molecular testing of patients with rare Rh phenotypes.

ABO Blood-Group System↗
PubMed2006

Molecular characterization of GYPB and RH in donors in the American Rare Donor Program.

Transfusion of patients with sickle cell disease (SCD) has been a challenge in clinical transfusion medicine, especially when the required donor RBCs must be U- and negative for high-prevalence Rh phenotypes (hr(B), hr(S)). It is now possible to genotype donors to identify or confirm Uvar and U- phenotypes, as well as Rh hr(B)- and hrS- phenotypes, and to characterize the different RH backgrounds found in these donors. In a preliminary study of donors registered in the American Rare Donor Program, twelve different RH backgrounds were identified in eighteen hr(B)- or hr(S)- donors. These results, summarized in the current report, confirm the heterogeneous nature of these phenotypes and are relevant for selection of donor units for patients with antibodies to high-prevalence Rh antigens. Not all phenotypically similar units will be compatible, and matching the Rh genotype of the donor to the patient is important to prevent further Rh sensitization. Most donors referred were hr(B)- and carry at least one hybrid RHD-CE(3-7)-D gene that encodes a variant C antigen linked to RHCE*ceS that encodes the VS+V- phenotype. Surprisingly, the majority of donors were heterozygous, some even carrying conventional alleles, suggesting that the loss of expression of the hr(B) epitopes on RBCs is a dominant phenotype. Although antigen-matching of patients with SCD with donors for C, E, and K antigens has decreased the incidence of alloimmunization, some patients still become immunized to Rh antigens, indicating the units were not truly matched. RH genotyping can identify those patients with SCD who carry RH alleles that encode altered C, e, or D who are at risk for production of "apparent auto" and alloantibodies to Rh antigens. RH genotyping of alloimmunized patients with SCD, partnered with genotyping of donors, can identify compatible units that would also eliminate the risk of further Rh alloimmunization.

Anemia, Sickle Cell↗
PubMed2004

Review: the Kell, Duffy, and Kidd blood group systems.

After the discovery (over 50 years ago) that the IAT could be applied to the detection of antibodies to blood group antigens, there was a rapid increase in the identification of alloantibodies that caused transfusion reactions or HDN. After Rh, antibodies in the Kell, Duffy, and Kidd blood group systems were the next in clinically significant antibodies to be revealed. Much of what has been learned about these blood groups since the journal Immunohematology issued its first edition has to do with the proteins, the genes, and the molecular basis for the antigens. What has not changed is that, after ABO and Rh, antibodies to antigens in these three systems are still the most clinically significant. They will form the basis of this review.

Amino Acid Substitution↗
PubMed2003

DNA analysis for donor screening of Dombrock blood group antigens.

Due to the scarcity of reliable antibodies, RBC typing for Doa and Dob is notoriously difficult. Inaccurate typing can place patients at risk for hemolytic transfusion reactions. The molecular basis of the DOA/DOB polymorphism is associated with three nucleotide changes:378 C>T, 624 T>C,and 793 A>G of DO. While the 378 C>T and 624 T>C are silent mutations, the 793A>G polymorphism in codon 265 encodes asparagine for Doa and aspartic acid for Dob. We describe here the use of a PCR-RFLP assay as an alternative to traditional hemagglutination for typing donor blood for Dombrock. Primers were designed to amplify the region of DO containing the 793A>G polymorphism. DNA samples from blood donors were amplified and subjected to RFLP analysis. A total of 613 samples were tested for the Dombrock polymorphism (793 A>G) by PCRRFLP. PCR-RFLP can be used to screen for Do(a-) or Do(b-) donors. This approach overcomes the scarcity of the reagents required for testing by hemagglutination.

Journal Article↗
PubMed2001

16Cys encoded by the RHce gene is associated with altered expression of the e antigen and is frequent in the R0 haplotype.

Serological observations have suggested that numerous D, many e (especially in Blacks), several E, and rare c variants exist within the Rh blood group system. The molecular basis for expression of many of these variants has been elucidated. This study describes five unrelated Caucasians whose red blood cells reacted with polyclonal anti-e but did not react with some monoclonal anti-e, which suggested that they carried a variant e antigen. Molecular investigation revealed the presence of a 48G-->C change (encoding cysteine instead of tryptophan at amino acid 16) in their RHce gene. No other differences were found, which suggests that amino acid residues located in the first transmembrane region can affect expression of the e antigen, whose critical residues are on the predicted fourth external loop of the protein. This polymorphism has not previously been observed because polyclonal anti-e does not distinguish this variant from wild type. This position is polymorphic in RHce alleles and the presence of the 48C nucleotide is often found in the R0 (Dce) haplotype.

Antibodies, Monoclonal↗
PubMed2000

Evidence supporting the requirement for two proline residues for expression of c.

BACKGROUND: In humans, c antigen expression is associated with a proline residue at amino acid position 103 in the second extracellular loop of the CE protein. Comparison of nonhuman primate Rh proteins suggested that c reactivity might actually involve two proline residues. It has been shown that the RBCs of New World capuchin monkeys (Cebus apella) react with anti-c. To further define the amino acid residues involved in c expression, Rh cDNA from the capuchin was analyzed. STUDY DESIGN AND METHODS: Rh transcripts were amplified by reverse transcription PCR from RNA isolated from the reticulocytes of a capuchin monkey and were cloned and sequenced. RESULTS: Rh transcripts from the capuchin monkey, whose RBCs react with anti-c, were found to encode adjacent proline residues at 102 and 103. CONCLUSION: Sequencing of Rh transcripts from the capuchin monkey supports the hypothesis that the expression of c requires two adjacent proline residues. Proline causes bends or loops in proteins, which, in this case, might form a unique, stable structure resistant to perturbations induced by changes in upstream or downstream residues. This would explain the scarcity in humans of c variants as compared to the other major Rh antigen variants, and the preservation of c reactivity despite 24-percent divergence between the human and capuchin Rh proteins.

Animals↗
PubMed1999

Unusual amino acid usage in the variable regions of mercury-binding antibodies.

Monoclonal antibodies (mAb) specific for mercuric ions were isolated from BALB/c mice injected with a mercury-containing, hapten-carrier complex. The antibodies reacted by enzyme-linked immunosorbent assay with bovine serum albumin-glutathione-mercuric chloride (BSA-GSH-HgCl) but not with BSA-GSH without mercury. Nucleotide sequences from polymerase chain reaction products encoding six of the antibody heavy-chain variable regions and seven light-chain variable regions revealed that all the antibodies contained an unpaired cysteine residue in one hypervariable region, which is unusual for murine antibodies. Mutagenesis of the cysteine to either tyrosine or serine in one of the Hg-binding antibodies, mAb 4A10, eliminated mercury binding. However, of two influenza-specific antibodies that contain cysteine residues at the same position as mAb 4A10, one reacted with mercury, although not so strongly as 4A10, whereas the other did not react at all. These results suggested that, in addition to an unpaired cysteine, there are other structural features, not yet identified, that are important for creating an appropriate environment for mercury binding. The antibodies described here could be useful for investigating mechanisms of metal-protein interactions and for characterizing antibody responses to structurally simple haptens.

Amino Acid Sequence↗
PubMed1999

Characterization of the mouse Rh blood group gene.

The Rh blood group system is of clinical importance in blood transfusion and as the cause of hemolytic disease of the newborn. Other than their role as carriers of Rh antigens, very little is known about the function of the Rh polypeptides. As a first step to generate an animal model system in which to study the structure and function of Rh, and to extend the phylogenetic analysis of RH genes, the Rh homologue from Mus musculus was characterized. Comparison of RH from humans and mice revealed 71 and 58% sequence identity at the nucleotide and amino acid levels, respectively. Mouse Rh mRNA encodes a protein which is 1 amino acid longer (418 aa) than that of human (417 aa). Rh protein was detected in mouse erythrocyte membranes and was comparable in size to human Rh. Mouse erythrocytes do not show serologic reactivity with human Rh antibodies, probably because the greatest divergence between the mouse and the human genes was seen in the predicted extracellular loops, while the transmembrane regions were more conserved. The mouse RH locus consists of only one gene, which is important for future genetic manipulation and which also indicates that the RH gene duplication seen in humans has occurred since the mammalian radiation.

Amino Acid Sequence↗
PubMed1997

DNA-binding antibodies from viable motheaten mutant mice: implications for B cell tolerance.

C57BL/6 me(v)/me(v) (viable motheaten) mice have defective hemopoietic cell protein-tyrosine phosphatase (HCP, SHP-1) and are a model in which to study the role of this phosphatase in immune regulation and hemopoietic development. Immune defects in these mice include hypergammaglobulinemia, production of multiple autoantibodies, and glomerular nephritis. The presence of self-reactive Abs in these mice is an enigma, since it has been demonstrated that they have increased negative selection against self-reactive transgenic Abs. It was of interest to examine autoantibodies from these mice to identify reactivity or structural features that might account for their persistence and expansion in the repertoire. Abs to DNA were selected for investigation, since they have been characterized extensively from normal and autoimmune strains of mice. Viable motheaten mice had high titers of IgM, anti-dsDNA Abs, and all 12 anti-DNA hybridomas generated were also IgM. They had high avidity for dsDNA, also bound to ssDNA, but were not polyreactive. They used V(H) and V(L) gene families found in anti-DNA Abs from other strains, which reinforces evidence that there is preferential use of a restricted number of germline elements in anti-DNA Abs. The results indicate that Hcph-mutant mice do not delete DNA-reactive B cells, nor are they able to maintain these B cells in a state of anergy. Evidence for positive selection and clonal expansion after V-D-J rearrangement was also seen, in that several Abs expressed the same heavy chain in combination with different light chains, which is also a hallmark of the autoimmune anti-DNA response.

Amino Acid Sequence↗
PubMed1996

Metal binding properties of a monoclonal antibody directed toward metal-chelate complexes.

A monoclonal antibody that recognizes cadmium-EDTA complexes has been produced by the injection of BALB/c mice with a metal-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to recognize 16 different metal-EDTA complexes was assessed by measuring equilibrium binding constants using a KinExATM immunoassay instrument. The antibody bound to cadmium- and mercury-EDTA complexes with equilibrium dissociation constants of 21 and 26 nM, respectively. All other metal-EDTA complexes tested, including those of Mn(II), In(III), Ni(II), Zn(II), Co(II), Cu(II), Ag(I), Fe(III), Pb(II), Au(III), Tb(III), Ga(III), Mg(II), and Al(III) bound with affinities from 20- to 40,000-fold less than that determined for the cadmium-EDTA complex. With the exception of mercury and magnesium, the binding of divalent metal-chelate complexes was well-correlated with the size of the metal ion. The amino acid sequences of the heavy and light chain variable regions were deduced from polymerase chain reaction-amplified regions of the corresponding genes and subsequently used to construct molecular models of the antigen binding region. The key residue for cadmium binding in the model for 2A81G5 appeared to be histidine 96 in the heavy chain.

Amino Acid Sequence↗
PubMed1996

Investigation of the RH locus in gorillas and chimpanzees.

The human Rh blood-group system is encoded by two homologous genes, RhD and RhCE. The RH genes in gorillas and chimpanzees were investigated to delineate the phylogeny of the human RH genes. Southern blot analysis with an exon 7-specific probe suggested that gorillas have more than two RH genes, as has recently been reported for chimpanzees. Exon 7 was well conserved between humans, gorillas, and chimpanzees, although the exon 7 nucleotide sequences from gorillas were more similar to the human D gene, whereas the nucleotide sequences of this exon in chimpanzees were more similar to the human CE gene. The intron between exon 4 and exon 5 is polymorphic and can be used to distinguish the human D gene from the CE gene. Nucleotide sequencing revealed that the basis for the intron polymorphism is an Alu element in CE which is not present in the D gene. Examination of gorilla and chimpanzee genomic DNA for this intron polymorphism demonstrated that the D intron was present in all the chimpanzees and in all but one gorilla. The CE intron was found in three of six gorillas, but in none of the seven chimpanzees. Sequence data suggested that the Alu element might have previously been present in the chimpanzee RH genes but was eliminated by excision or recombination. Conservation of the RhD gene was also apparent from the complete identity between the 3'-noncoding region of the human D cDNA and a gorilla genomic clone, including an Alu element which is present in both species. The data suggest that at least two RH genes were present in a common ancestor of humans, chimpanzees, and gorillas, and that additional RH gene duplication has taken place in gorillas and chimpanzees. The RhCE gene appears to have diverged more than RhD among primates. In addition, the RhD gene deletion associated with the Rh-negative phenotype in humans seems to have occurred after speciation.

Amino Acid Sequence↗
PubMed1994

Investigation of the human Rh blood group system in nonhuman primates and other species with serologic and Southern blot analysis.

To investigate the evolution of the Rh blood-group system in anthropoid apes, New and Old World monkeys, and nonprimate animals, serologic typing of erythrocytes from these species with antibodies specific for the human Rh blood-group antigens was performed. In addition, genomic DNA from these animals was analyzed on Southern blots with a human Rh-specific cDNA. Consistent with earlier reports, serologic results showed that gorilla and chimpanzee erythrocytes had epitopes recognized by human Rh D and c antisera, and gibbon erythrocytes were recognized by the c antisera. Surprisingly, some Old and New World monkeys also expressed a Rh c epitope on their erythrocytes. No erythrocytes from the nonprimate animals reacted specifically with any of the human Rh antisera. Southern blot analysis with a human Rh-specific cDNA probe detected Rh-related sequences in anthropoid apes, all New and Old World monkeys, and in most nonprimate animals tested. Although some Rh-related restriction fragments were conserved across species lines in primates, the Rh locus was more polymorphic in chimpanzees and gorillas than in humans. In addition, restriction fragments segregating with the presence of the D antigen in humans were present in the primate species that expressed the D antigen.

Animals↗
PubMed1994

A comparison of the reactivity of monoclonal and polyclonal Rh reagents with red cells after prolonged storage.

Previous studies with stored red cells collected into EDTA anticoagulant had shown that commercial polyclonal anti-C, -c, and -E reagents gave acceptable reactions for 60 days, hut polyclonal anti- e reagents reliably detected the e antigen only through 14 days. At that time it was noted that a monoclonal anti-e reacted 3+ to 4+ with red cells that no longer reacted, or reacted very weakly, with the polyclonal anti-e reagents. This observation led to a comparison study of the reactivity of monoclonal versus polyclonal R h reagents with stored red cells. Monoclonal and polyclonal anti-C, -c, and -E reagents showed comparable reaction strengths, and gave acceptable reactions over the entire testing period. Polyclonal anti-e reagents, which are known to he of lower titer, again did not give reliable reactions with stored cells after 14 days. However, three different monoclonal anti-e reagents were able to detect the presence of the e antigen on stored cells for many weeks, This study showed that the monoclonal anti-e reagents tested are superior to polyclonal anti-e reagents for typing specimens that have been stored for more than 14 days.

Journal Article↗
PubMed1993

Red cell antigen stability in K3EDTA.

Commercial blood grouping reagents are not approved for testing EDTA anticoagulated blood specimens that are more than 48 hours old. Many studies on the stability of blood group antigens in other anticoagulants have been reported, but none are available for EDTA. This study was undertaken to assess whether current commercially available blood grouping reagents give acceptable reactions with red cell antigens when the cells are stored for mended periods in EDTA We defined acceptable reaction strength to be 2+ (score 8). As expected, the A, B, and D reactions were very stable with red cells stored for 60 days. All antigens except Lea exhibited 2+ (score 8) or greater reactions at day 14, and at day 2 1 only the Lea, Fyb, and e antigens were less than 2+. On day 60, twelve of twenty-one antigens tested still exhibited 2+ or greater reactions. This study shows that antigen reactivity for red cells collected and stored in EDTA is at least equal to that for clotted specimens These red cells can be used for reliable antigen typing for at least 14 days, and even longer for most antigens.

Journal Article↗