Biomedical subjects

R M Kaufman

Publications and source records attributed to R M Kaufman.

11 recordsLinked to original sources

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↗

PubMed2001

Lack of neighborhood effects from a transcriptionally active phosphoglycerate kinase-neo cassette located between the murine beta-major and beta-minor globin genes.

For the treatment of beta-globin gene defects, a homologous recombination-mediated gene correction approach would provide advantages over random integration-based gene therapy strategies. However, "neighborhood effects" from retained selectable marker genes in the targeted locus are among the key issues that must be taken into consideration for any attempt to use this strategy for gene correction. An Ala-to-Ile mutation was created in the beta6 position of the mouse beta-major globin gene (beta(6I)) as a step toward the development of a murine model system that could serve as a platform for therapeutic gene correction studies. The marked beta-major gene can be tracked at the level of DNA, RNA, and protein, allowing investigation of the impact of a retained phosphoglycerate kinase (PGK)-neo cassette located between the mutant beta-major and beta-minor globin genes on expression of these 2 neighboring genes. Although the PGK-neo cassette was expressed at high levels in adult erythroid cells, the abundance of the beta(6I) mRNA was indistinguishable from that of the wild-type counterpart in bone marrow cells. Similarly, the output from the beta-minor globin gene was also normal. Therefore, in this specific location, the retained, transcriptionally active PGK-neo cassette does not disrupt the regulated expression of the adult beta-globin genes. (Blood. 2001;98:65-73)

Animals↗

PubMed1999

Transgenic analysis of a 100-kb human beta-globin cluster-containing DNA fragment propagated as a bacterial artificial chromosome.

To date, the normal transcriptional regulation of the human beta-globin gene cluster has been recapitulated most accurately in transgenic mice that carry large yeast artificial chromosome (YAC) or ligated cosmid constructs. However, these large transgenes still exhibit variegated expression levels, perhaps because they tend to rearrange upon integration, or because the cloning vectors remain attached to the globin inserts. To try to circumvent these potential problems, we investigated the transgenic properties of a 100-kb DNA fragment containing the entire human beta-globin cluster propagated in a bacterial artificial chromosome (BAC). We created 9 independent mouse lines, each carrying 1 to 6 copies of the human beta-globin cluster without the attached BAC vector. Five of the lines carry unrearranged copies of the cluster. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis of adult F(1) mice showed that 2 lines express human beta globin at levels approximately equivalent to the endogenous mouse beta-major genes. One line expresses no human beta globin, while the remaining 6 lines show intermediate expression levels. Complete gamma-->beta-globin gene switching occurs, but is slightly delayed with respect to the endogenous mouse embryonic-->adult switch. Since these data are similar to what has been obtained using globin YACs or ligated cosmids, we conclude that (1) globin transgenes propagated in BACs are no less likely to rearrange than their cosmid or YAC counterparts, and (2) the retention of YAC vector sequences in a transgene probably has no significant impact on globin expression when using constructs of this size.

Animals↗

PubMed1995

Identification of complement regulatory domains in human factor H.

Factor H, a regulator of complement activation, contains 20 short consensus repeat (SCR) domains common among the family of C3b/C4b-binding proteins. Chinese hamster ovary cells transfected with cDNA corresponding to the N-terminal tryptic fragment of factor H (containing SCR 1-5 and part of SCR 6) secreted protein with cofactor activity for factor I-dependent cleavage of C3b. A series of deletion mutants, each lacking one of the first five SCR, were constructed, and the supernatants of transfected Chinese hamster ovary cells were tested for cofactor activity. Supernatants of Chinese hamster ovary cells transfected with SCR 1, SCR 4, and SCR 5 deletion mutants retained cofactor function, although the SCR 1 deletion had reduced cofactor activity. Deletion of SCR 2 or 3 totally abolished cofactor activity. Expression and functional analysis of SCR units 1-3, 2-3, and 2-4 demonstrated that the SCR 1-3 unit is sufficient for cofactor activity, but SCR 1-4 is required for full activity. For assays involving cell protection, a construct linking SCR 1-5 to the glycosyl-phosphatidylinositol anchor of decay-accelerating factor was prepared, and stable transfectants were obtained. These cells were protected against complement-mediated cytotoxicity, similarly to decay-accelerating factor- and membrane cofactor protein-transfected cells. These studies define the complement regulatory domains in factor H and suggest that the general complement functional unit for C3 convertase regulation involves three or four consecutive SCR units.

Animals↗

PubMed1994

Molecular mapping of the Cromer blood group Cra and Tca epitopes of decay accelerating factor: toward the use of recombinant antigens in immunohematology.

Cromer blood group antigens reside on the complement regulatory protein decay accelerating factor (DAF, CD55). This glycosyl-phosphatidylinositol-anchored glycoprotein is widely distributed, especially among cell types in contact with plasma. Numerous Cromer blood group antigens have been defined using alloantibodies induced by transfusion or pregnancy. However, few pairs of antithetical antigens have been described in this system, presumably because of the rarity of the low-frequency alleles. Analysis of polymerase chain reaction-amplified genomic DNA showed that the Cr(a-) phenotype has a Ala193-->Pro substitution in short consensus repeat 4 (SCR4) of DAF, and the Tc(a-b+) phenotype has a Arg18-->Leu substitution in SCR1 of DAF. The locations of Cra and Tca epitopes were confirmed by analysis of Chinese hamster ovary cell transfectants expressing a Cr(a-) allele-specific transfectant and a chimeric protein containing only SCR1 of DAF, respectively. Overall, these studies further show the usefulness of an approach based on recombinant proteins in mapping blood group antigen epitopes and identifying blood group antibodies.

Antigens, CD↗

PubMed1971