Clinical problem-solving: costly errors.
Explore the source record for details and available documents.
Anemia, Hemolytic, Autoimmune↗
Biomedical subjects
M J Telen
Publications and source records attributed to M J Telen.
Erythrocyte blood group antigens: not so simple after all.
Explore the source record for details and available documents.
Animals↗
Blood group antigens on complement receptor/regulatory proteins.
Explore the source record for details and available documents.
Antigens, CD↗
Glycosyl phosphatidylinositol-linked blood group antigens and paroxysmal nocturnal hemoglobinuria.
Human erythrocyte cell surface molecules that are attached to the cell membrane by glycosyl-phosphatidylinositol (GPI) anchors include the complement regulatory proteins decay accelerating factor (DAF, CD55) and membrane inhibitor of reactive lysis (MIRL, CD59), as well as the proteins that bear the Cartwright, Dombrock, and JMH blood group antigens. The acquired hematopoietic stem cell disorder paroxysmal nocturnal hemoglobinuria (PNH) results from the absence or marked deficiency in expression of GPI-anchored proteins in affected hematopoietic cells. PNH usually if not always results from a somatic mutation of an X-linked gene called PIG-A; the product of the PIG-A gene is a glycosyl transferase necessary for construction of the GPI anchor. DAF is a ubiquitously expressed protein present in many tissues, including gastrointestinal epithelia, corneal epithelia, and serosa of urinary and reproductive organs. DAF is a 70 kD glycoprotein containing complement regulatory short consensus repeats (SCRs); its gene is located in the regulation of complement activation (RCA) gene cluster on chromosome 1 and is about 40 kb in size. The Cromer blood group antigens, which reside on DAF, include 10 currently defined antigens, of which seven are of high incidence. The molecular basis of the Cr (a-) phenotype has been determined to be a single base pair substitution in DAF SCR4 (G-->C, leading to an ala193 to pro amino acid substitution). The Tc alpha antigen appears to be determined by the amino acid sequence of SCR1, with the Tc (a-b+) phenotype arising from a base pair substitution of G55-->T, leading to an arg18 to leu amino acid substitution. The null phenotype for Cromer antigens occurs when DAF is completely absent; only one example has been completely studied on the molecular level. That individual is homozygous for a point mutation in SCR1 (G314-->A) that creates a stop codon (TGA) in place of one normally encoding trp53 (TGG) and thus prevents further translation of the mRNA. The Dr(a-) phenotype expresses reduced quantities of DAF (approximately 40% of normal levels), as well as a polymorphism of DAF. Lack of the Dr alpha antigen has been proved to result from a single point mutation in SCR3 (C-->T in codon 165) that leads to a single amino acid substitution (ser-->leu). The Cartwright (Yt) antigens reside on acetylcholinesterase (AChE). In erythroid cells, a small exon that encodes the signal for attachment of the GPI anchor is retained in a tissue-specific process.(ABSTRACT TRUNCATED AT 400 WORDS)
Acetylcholinesterase↗
Lutheran antigens, CD44-related antigens, and Lutheran regulatory genes.
The Lutheran (Lu) blood group antigens are a family of human erythrocyte antigens which reside on two closely-related erythrocyte integral membrane proteins. Sixteen Lutheran or so-called para-Lutheran antigens have thus far been described, and human antisera to many of them have been shown to immunoblot two proteins, of 78 and 85 kDa. Lu cDNA encodes an integral membrane protein of 597 amino acids that is a member of the Ig superfamily. Lu proteins comprise five Ig superfamily domains, along with a single transmembrane domain and a cytoplasmic domain of about 60 amino acids. The two proteins seen in biochemical studies of red cell membranes appear to be derived from 2 mRNA species that differ only in their 3' ends, suggesting that they arise from alternate splicing of a single preRNA. Three genetic backgrounds for the Lu(a-b-) [Lu null] phenotype have been described. A recessive Lu null phenotype is rarely observed as a result of homozygosity for two amorphic LU alleles. However, the most common Lu(a-b-) phenotype appears to be caused by an independently segregating, dominant gene, designated In (Lu), which inhibits expression of all Lutheran antigens to nearly undetectable levels. This gene also affects the expression of other cell surface proteins and blood group antigens that are genetically unlinked to the Lutheran locus, including CD44 and MER2. CD44, a member of the cartilage link family of proteins, bears the In and AnWj blood group antigens. A widely distributed protein CD44 is expressed at normal levels on all tissues except erythrocytes in the presence of the In (Lu) gene. A second Lutheran regulatory gene, XS2, is responsible for the third Lu(a-b-) phenotype, which exhibits an X-linked inheritance pattern. The XS2 gene down-regulates but does not abolish expression of LU genes and does not affect expression of CD44.
DNA, Complementary↗
Location of WESb on decay-accelerating factor.
Explore the source record for details and available documents.
Antigens↗
Investigations using a novel monoclonal antibody to the glycosylphosphatidylinositol-anchored protein that carries Gregory, Holley, and Dombrock blood group antigens.
BACKGROUND: The high-frequency Hy and Gya antigens have been shown to reside on the same protein. Gy(a-) Hy-negative red cells are also Do(a-b-). A mouse monoclonal antibody, 5B10, was produced with specificity related to the human Gregory, Holley, and Dombrock blood group antigens. STUDY DESIGN AND METHODS: The antibody reacted in direct hemagglutination assays, and its specificity was investigated by radioimmunoassay, inhibition assay, and Western blotting. RESULTS: The 5B10 antibody failed to bind to abnormal paroxysmal nocturnal hemoglobinuria red cells and human erythroleukemia cell line K562, but it was weakly reactive with HEL cells. Red cells, but not other circulating hematopoietic cells, express the 5B10 antigen. The 5B10 antibody had a specificity similar but not identical to that of Gya. Gy(a-) Hy-negative red cells reacted extremely weakly with 5B10 antibody, but Gy(a-) Hy-negative red cells treated with a variety of proteases bound 5B10 antibody strongly. This suggests that these cells express a variant form of the protein recognized by 5B10. CONCLUSION: Identification of a monoclonal antibody to this glycosylphosphatidylinositol-linked protein opens a new avenue for investigation of the biochemistry, genetics, and function of the glycosylphosphatidylinositol-linked protein that bears the Gya, Hy, and Do antigens.
Alleles↗
Evidence that CDw108 membrane protein bears the JMH blood group antigen.
BACKGROUND: CDw108 is a cluster-of-differentiation antigen that resides on a glycosylphosphatidylinositol (GPI)-linked protein; it has not previously been shown to be expressed on red cells. JMH is a high-frequency red cell blood group antigen that resides on a GPI-linked protein of molecular weight similar to that bearing CDw108. The purpose of this study was to investigate whether CDw108 is expressed on red cells and whether it resides on the same membrane protein as does JMH. STUDY DESIGN AND METHODS: Murine monoclonal antibodies to CDw108, MEM-121 and MEM-150, as well as a murine monoclonal antibody and human antibodies to JMH were used in radioimmunoassay, inhibition assay, Western blotting, and monoclonal antibody-specific immobilization of erythrocyte antigen assay. RESULTS: MEM-121 and MEM-150 were found to bind to red cells, and MEM-150 blocked binding of human anti-JMH to red cells. Anti-CDw108 and anti-JMH identified red cell membrane proteins that were of similar size and that were absent from JMH-negative red cells on Western blotting. MEM-150 and MEM-121 also immobilized the same protein that reacted with human anti-JMH. CONCLUSION: CDw108 is expressed on red cells and resides on the same GPI-linked membrane protein as does the JMH blood group antigen.
Antibodies, Monoclonal↗
Identification of the Tcb allele of the Cromer blood group gene by PCR and RFLP analysis.
The Cromer blood group antigens reside on the complement regulatory protein, decay-accelerating factor (DAF). The Cromer system comprises 10 antigens, 3 of which are of low incidence. When an individual is homozygous for the allele encoding one of these low-incidence antigens, they are liable to produce an antibody to the antithetical high-frequency antigen if challenged by pregnancy or transfusion. These antibodies are often difficult to identify, because of the lack of readily available antigen-negative cells and typing sera. In blacks, about 5 percent of individuals carry the rare Tcb Cromer allele. We have shown that the presence of the low-incidence Tcb allele can be detected by polymerase chain reaction (PCR) amplification of a fragment of the gene encoding DAF, followed by allele-specific restriction enzyme digestion.
Journal Article↗
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↗
Molecular basis of reduced or absent expression of decay-accelerating factor in Cromer blood group phenotypes.
The human erythrocyte blood group system Cromer consists of high-incidence and low-incidence antigens that reside on decay-accelerating factor (DAF; CD55), a glycosyl-phosphatidylinositol-anchored membrane protein that regulates complement activation on cell surfaces. In the Cromer phenotypes Dr(a-) and Inab there is reduced or absent expression of DAF, respectively. This study investigated the molecular basis of the reduced DAF expression by polymerase chain reaction amplification of genomic DNA and RNA/cDNA obtained from Epstein-Barr virus-transformed lymphoblastoid cell lines. Sequence analysis of the Inab propositus showed a single nucleotide substitution in exon 2 of the DAF gene and at the corresponding position in the cDNA, G314-->A resulting in Trp53-->Stop. This truncation near the amino terminus explains the complete absence of surface DAF in the Inab phenotype. A similar analysis was performed for two Dr(a-) individuals, including KZ, who was previously reported to be Inab phenotype but is now shown by immunochemical and serologic methods to be Dr(a-) phenotype. A single nucleotide change was found in exon 5 of the DAF gene, C649-->T resulting in Ser165-->Leu, which we had previously shown to lead to loss of the Dra epitope. However, two species of cDNA were found, one encoding full-length DAF with the single amino acid change and the more abundant species having a 44-nucleotide deletion. The 44 nucleotide deletion includes the single polymorphic site, which creates a cryptic branch point in the Dr(a-) allele that leads to use of a downstream cryptic acceptor splice site. This shifts the reading frame and leads to a premature stop codon that precludes membrane anchoring. Thus, the single point mutation in the Dr(a-) phenotype results in a novel use of alternative splicing and provides a molecular explanation for both the antigenicity and the reduced DAF expression seen in this phenotype.
Amino Acid Sequence↗
Rh-related antigen CD47 is the signal-transducer integrin-associated protein.
Integrin-associated protein (IAP) is a 50-kDa membrane protein with an amino-terminal immunoglobulin domain and a carboxyl-terminal multiply membrane-spanning region. It is physically and functionally associated with the integrin alpha v beta 3 vitronectin receptor and is involved in the increase in intracellular calcium concentration, which occurs upon cell adhesion to extracellular matrix. Oxidative burst in neutrophils can be induced or inhibited via IAP. Surprisingly, IAP is also expressed on erythrocytes, which have no known integrins. IAP has been shown to be identical to OA3, an ovarian carcinoma antigen. We now show that IAP expression is reduced on Rhnull erythrocytes. The IAP structural gene is mapped to q13.1-2 on human chromosome 3, within a region known to contain a gene encoding the Rh-associated 1D8 antigen. By expression studies on human erythrocytes and IAP transfectants, IAP is shown to be identical to the 1D8 antigen and to CD47, a cell surface protein with broad tissue distribution, reduced in expression on Rhnull erythrocytes. Two CD47 antibodies recognize the immunoglobulin domain of IAP, as does antibody 1D8. These studies suggest the possibility that IAP and the Rh polypeptides may share a pathway for membrane expression on erythrocytes. Furthermore, decreased expression of IAP on Rhnull cells may contribute to the these cells' abnormal cation permeabilities. These studies demonstrate an unexpected link between integrin signal transduction and erythrocyte membrane structure.
Animals↗
Expression of the cell adhesion molecule CD44 in gastric adenocarcinomas.
CD44, an integral membrane glycoprotein expressed by many cell types, serves as the principal transmembrane hyaluronate receptor and may be a determinant of metastatic and invasive behavior in carcinomas. The expression of CD44 in 23 gastric adenocarcinoma and 12 peptic ulcer disease (PUD) resection specimens and gastric carcinoma cell lines HS746t and KATO III was examined by immunohistochemistry using the murine monoclonal antibody A3D8 on formalin-fixed, paraffin-embedded tissue or cells. Western blot analysis of whole cell lysates of KATO III and HS746t cells showed protein bands at 85 to 90 kd with KATO III cells expressing an additional band at 145 kd. In normal stomach gastric epithelium was negative. In PUD foveolar epithelium was focally positive, but staining did not correlate with the extent of gastritis. In carcinoma cases intensity of staining was progressively stronger comparing intestinal metaplasia with dysplasia with intramucosal carcinoma. Invasive carcinoma was invariably more strongly positive than dysplasia or intramucosal carcinoma. Twelve adenocarcinomas were weakly positive and 11 were strongly positive. The staining intensity of metastases (12 cases) was the same or weaker than the primary tumor. For the 12 patients whose carcinomas were weakly positive, mean length of survival for the six who died was 23.3 months. Five of the 11 patients whose carcinomas strongly expressed CD44 died within the study period with a mean length of survival of 11.0 months. A key consequence of CD44 overexpression in gastric carcinomas may be development of the invasive phenotype and strong expression may indicate a poorer prognosis.
Adenocarcinoma↗
Tissue culture of epithelium derived from Barrett's oesophagus.
Barrett's oesophagus is a preneoplastic condition in which the squamous mucosa of the oesophagus is replaced by columnar epithelium. Epithelial cells of Barrett's oesophagus were isolated from resected oesophagus specimens by two methods not previously applied to the culture of Barrett's oesophagus cells. These techniques included trypsinisation of small fragments of mucosa, followed by plating in tissue culture dishes, and a direct tissue explant technique. A modified MCDB-153 growth medium was used. Primary trypsin technique cultures were plated on uncoated plastic, or plastic coated with type I collagen, type IV collagen, or fibronectin. Growth on type IV collagen and fibronectin plates was slower but produced less contamination from fibroblasts. By 20-40 days most cultures formed confluent monolayers made up of cells with epithelial morphology. The cells were cytokeratin positive, vimentin negative, and contained alcian blue positive vacuoles, confirming their epithelial origin and suggesting their derivation from Barrett's oesophagus. Electron microscopy showed tonofilaments, microvilli, and desmosomes. Cells proliferated through up to eight subcultures before growth slowed and cells showed senescent changes. This study shows that epithelial cells from Barrett's oesophagus can be grown by comparatively simple tissue culture techniques. These methods can provide sufficient material for a variety of molecular biology and biochemical studies of epithelial cells from Barrett's oesophagus.
Barrett Esophagus↗
Recent advances in immunohematology.
Knowledge of the biochemistry and genetics of erythrocyte blood group antigens has been growing rapidly over the past several years. Last year, the molecular basis for the major Rh blood group antigens was delineated. In addition, the genetic and biochemical bases of several other blood group antigens were identified. One of the most interesting matches of blood group antigens to a functional membrane protein was that of the Diego antigens to the erythrocyte anion channel (band 3) protein. In addition, knowledge of the molecular basis of blood group antigens is now leading rapidly to the usefulness of molecular techniques in identifying blood group genotypes and even blood group antibody specificities. This knowledge has also broadened our understanding of the pathogenesis of erythrocyte disorders associated with null blood group phenotypes. The diagnosis and treatment of autoimmune hemolytic anemia has seen slow but steady progress. New techniques appear promising as methods for distinguishing clinically important from benign autoantibodies. The molecular targets for erythrocyte autoantibodies were also largely identified. Treatment of autoimmune hemolytic anemia is also slowly being improved with the use of agents such as intravenous gammaglobulin, danazol, and immunosuppressive agents, all of which have also been important in the treatment of autoimmune thrombocytopenic purpura.
Anemia, Hemolytic, Autoimmune↗
Regulation of human CD44H and CD44E isoform binding to hyaluronan by phorbol myristate acetate and anti-CD44 monoclonal and polyclonal antibodies.
CD44 molecules are comprised of multiple alternatively spliced forms and are associated with diverse functions such as mediation of carcinoma metastasis and T cell coactivation. To study the function of individual CD44 isoforms, we have transfected CD44 isoforms into CD44-negative Jurkat T cells and produced cloned Jurkat cell lines that are stably transfected with either a CD44 isoform containing no alternatively spliced insert (CD44H) or a CD44 variant (CD44E) containing an insert of 132 amino acids derived from exons 12, 13, and 14 of the CD44 gene. We found that neither CD44H- nor CD44E-transfected Jurkat T cells constitutively bound hyaluronan (HA), whereas PMA treatment induced Jurkat cells transfected with CD44H but not CD44E to bind HA. CD44 mAb against noninsert regions of the CD44 extracellular domain (A3D8, A1G3) and polyclonal antisera against the COOH-terminal extracellular glycosaminoglycan region of CD44H (anti-6A serum) both induced CD44H-transfected cells to bind HA, whereas only one CD44 mAb (A1G3) induced CD44E-transfected Jurkat T cells to bind HA. Studies of Jurkat cells transfected with CD44H forms with truncations of the CD44 cytoplasmic domain demonstrated that the cytoplasmic COOH-terminal 52 amino acids were critical for binding of HA to the CD44 extracellular domain. Thus, these data underscore the importance of the CD44 cytoplasmic domain in the function of the extracellular portion of CD44H, and demonstrate a role for ligation of human CD44 isoforms at multiple distinct sites in regulation of expression of CD44 binding to HA.
Amino Acid Sequence↗
Human erythrocyte acetylcholinesterase bears the Yta blood group antigen and is reduced or absent in the Yt(a-b-) phenotype.
The Cartwright (Yt) blood group antigens have previously been shown likely to reside on a phosphatidylinositol-linked erythrocyte membrane protein. In this study, an unusual individual whose red blood cells (RBCs) were of the previously unreported Yt(a-b-) phenotype were used, along with normal Yt(a+) cells, to investigate serologically and biochemically the relationship of the Yta antigen to known phosphatidylinositol-linked erythrocyte proteins. Yt(a-b-) RBCs expressed normal amounts of various phosphatidyl-inositol-linked proteins except acetylcholinesterase. Further, human anti-Yta reacted with acetylcholinesterase in immunoprecipitation and immunoblotting studies. Thus, acetylcholinesterase is now identified as the protein bearing the Yt blood group antigens.
Acetylcholinesterase↗
Monoclonal antibody recognizing a unique Rh-related specificity.
A mouse IgG1 monoclonal antibody (MAb) UMRh, was prepared by immunizing Balb/c mice with the Jurkat T cell acute lymphoblastic leukemia (T-ALL) cell line. The MAb UMRh is directed against a widely distributed Rh-related cell surface antigen, present on red blood cells (RBCs) expressing the more common Rh phenotypes. The antigen has reduced expression on RBCs of -D-, DCW-/DCW-, Rhmod and Rhnull phenotypes. UMR immunoblotted a unique pattern on RBC membrane preparations of two bands at 40 and 43 kD and a diffuse pattern extending upward to about 55 kD. The UMRh antigen is also present on peripheral blood mononuclear cells, granulocytes, platelets, leukemic cells of T cell, B cell and myeloid origins, hematopoietic stem cells, and two tumor lines (lung and colon carcinoma). The number of UMRh sites per RBC (CDe/ce) was determined to be 5,519 copies/cell, whereas the sites on a -D- phenotype RBC were 1,096 copies/cell. A T-ALL line (CEM) expressed 333,364 copies/cell and a myeloid line (KG-1) expressed 90,913 copies/cell. Several Rh-related murine MAbs have been described, but our data indicates that UMRh recognizes a previously uncharacterized Rh-related specificity.
Animals↗