Makoto Uchikawa

Japanese Red Cross, Edo, Tōkyō, Japan

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Publications (53)159.2 Total impact

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    ABSTRACT: Background The high-prevalence antigen Jra is carried on the ATP-binding cassette transporter ABCG2. The ABCG2 gene consists of 16 exons and its translation start codon is located on the second exon. Although the occurrence of the Jr(a−) phenotype is rare, several ABCG2 null alleles have been reported. We report a new ABCG2 null allele having a large deletion in this study.Study Design and Methods The Jra status was determined by standard serologic tests and genomic DNA was isolated from whole blood. Exons 1 to 16 and the 5′-untranslated region of the ABCG2 gene were analyzed by polymerase chain reaction and sequencing. Expression of the ABCG2 protein on red blood cells was examined by immunoblotting.ResultsA Jr(a−) blood donor had a novel allele having a 27-kb deletion including noncoding Exon 1 and the promoter region of ABCG2, and the donor was apparently homozygous for the allele. In addition, we found three more individuals having heterozygosity for the same allele, with ABCG2*01N.01 having c.376C>T (p.Q126X), but did not find the allele having the 27-kb deletion in 3000 Jr(a+) individuals. Immunoblotting revealed that the ABCG2 protein was not found to be expressed in the individual with homozygosity for the ABCG2 27-kb deleted and in two individuals with an ABCG2 27-kb deleted/ABCG2*01N.01 genotype, which indirectly allows to conclude that the 27-kb deletion is responsible for a null ABCG2 allele.Conclusion We first identified an ABCG2 null allele (provisional ISBT allele number ABCG2*01N.23) having a large deletion including the promoter region.
    Transfusion 01/2015; · 3.53 Impact Factor
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    ABSTRACT: We developed a sequence-specific primer PCR (SSP-PCR) for detection of a 5·8-kb deletion (Bm5·8) involving an erythroid cell-specific regulatory element in intron 1 of the ABO blood group gene. Using this SSP-PCR, we performed genetic analysis of 382 individuals with Bm or ABm. The 5·8-kb deletion was found in 380 individuals, and disruption of the GATA motif in the regulatory element was found in one individual. Furthermore, a novel 3·0-kb deletion involving the element (Bm3·0) was demonstrated in the remaining individual. Comparisons of single-nucleotide polymorphisms and microsatellites in intron 1 between Bm5·8 and Bm3·0 suggested that these deletions occurred independently.
    Vox Sanguinis 11/2014; · 2.85 Impact Factor
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    ABSTRACT: Background and Objectives Previously, a weak phenotype Am or Bm was assumed to be caused by a reduction of A or B gene expression in bone marrow cells, but not in mucus-secreting cells. However, ABO expression has not been examined in erythroid progenitor cells of Am or Bm individuals.Materials and Methods We carried out in vitro erythroid differentiation of CD34+ cells from peripheral blood of a Bm individual harbouring a 3·0-kb deletion including an erythroid cell-specific regulatory element, named the +5·8-kb site, in intron 1 of the human ABO blood group gene.ResultsDuring the in vitro differentiation of CD34+ cells from this Bm individual into erythroid cells, B-antigens were not detectable on the cultured cells by flow cytometric analysis, and allele-specific RT-PCR consistently detected the transcripts from the O allele, but not from the B allele. Moreover, chromatin immunoprecipitation assay demonstrated that both RUNX1 and GATA-2 or GATA-1 were bound to the +5·8-kb site in cultured erythroid cells expressing ABO.Conclusion It is likely that the +5·8-kb site enhances transcription from the ABO promoter in erythroid cells through binding of RUNX1 and GATA-2 or GATA-1.
    Vox Sanguinis 11/2014; · 2.85 Impact Factor
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    ABSTRACT: Background and objectivesThe Kidd blood group system consists of polymorphic antigens, Jka (JK1) and Jkb (JK2), and a high-incidence antigen, Jk3. Anti-Jk3 is often observed in immunised Jk(a−b−) individuals. In this study, we aimed to establish a human hybridoma cell line secreting monoclonal anti-Jk3 (HIRO-294).Materials and methodsPeripheral blood lymphocytes of a Filipino woman with the Jk(a−b−) phenotype having anti-Jk3 were transformed with Epstein-Barr virus and then hybridised with the myeloma cell line JMS-3 using the polyethylene glycol (PEG) method. The reactivity and specificity of the anti-Jk3 were examined by serology and flow cytometry.ResultsFour hybridoma clones secreting anti-Jk3 were established and the antibody from one of these clones, HIRO-294, was examined. The reactivity of HIRO-294 was positive with 227 Jk(a+b−) red blood cells (RBCs), 298 Jk(a−b+) RBCs, and 1043 Jk(a+b+) RBCs, but was negative with 21 Jk(a−b−) RBCs. Eluates from Jk(a+b−) RBCs and Jk(a−b+) RBCs sensitised with the anti-Jk3 were cross-reacted with Jk(a−b+) RBCs and Jk(a+b−) RBCs, respectively. The reactivity of HIRO-294 was enhanced by the treatment of RBCs with ficin, trypsin, pronase and α-chymotrypsin, but was not changed by their treatment with neuraminidase, dithiothreitol and ethylenediaminetetraacetic acid (EDTA) glycine acid (GA). The RBCs sensitised by the anti-Jk3 were not agglutinated with the commercial reagents of anti-Jka and anti-Jkb by saline test, whereas the nonsensitised RBCs or those sensitised by monoclonal anti-D [HIRO-3, immunoglobulin G (IgG) class] were agglutinated with those reagents.Conclusions We established a human hybridoma cell line secreting monoclonal anti-Jk3 (HIRO-294). This antibody had unique specificity, recognising the Kidd glycoprotein including the Jka/Jkb polymorphic site.
    Transfusion Medicine 09/2014; · 1.26 Impact Factor
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    ABSTRACT: Background Transfusion-related acute lung injury (TRALI) is a life-threatening complication of blood transfusion. Antibodies against human leukocyte antigens in donors' plasma are the major causes of TRALI. Several animal models of TRALI have been developed, and the mechanism underlying TRALI development has been extensively investigated using rodent models. Although sheep models of nonimmune TRALI have been developed, large-animal models of antibody-mediated TRALI are not yet available.Study Design and Methods To develop a swine model of TRALI, male Clawn strain miniature pigs were used. A monoclonal antibody (MoAb) against swine leukocyte antigens (SLAs) Class I (4G8, 0.3 or 1.0 mg/kg body weight [BW]) and a control antibody (1.0 mg/kg BW) were injected into the peripheral vein after priming with or without 1 μg/kg BW lipopolysaccharide (LPS; n = 3 each). Lung injury was assessed using PaO2/FiO2 (P/F) ratio and by chest X-ray imaging. Histopathologic analysis was also conducted.ResultsLung injury could be induced by injecting 4G8 at an amount of 1.0 mg/kg BW, after LPS. The P/F ratio 90 minutes after the administration of 4G8 significantly decreased (p < 0.05). Bilateral infiltration was shown in chest X-ray imaging. Lung injury was confirmed by histopathologic analysis.Conclusion Lung injury in pigs was successfully induced by anti-SLA MoAb. Priming with LPS is a prerequisite for inducing lung injury and the amount of the antibody is a critical condition.
    Transfusion 07/2014; · 3.53 Impact Factor
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    ABSTRACT: Background and objectivesAn erythroid cell-specific regulatory element, referred to as the +5.8-kb site, has been identified in the first intron of the human ABO blood group gene. Subsequent studies have revealed involvement of deletion or mutation at the site in phenotypes Am, Bm and ABm. We investigated the molecular mechanisms involved in the A3 and B3 phenotypes. Materials and methodsGenomic DNAs were prepared from peripheral blood of seven A3 individuals and twelve B3 or AB3 individuals, and the nucleotide sequences were investigated using PCR and sequencing. Promoter assays were performed with K562 cells. ResultsTwo single point-mutations at +5893 or +5909 in the site on the A-allele were found in A3 individuals, while promoter assays revealed decreased activity at the site as a result of each substitution. In two B3 individuals, a single point-mutation at −77 in the ABO promoter on the B-allele was found, and the substitution was demonstrated to reduce the promoter activity. Conclusion Nucleotide substitutions in the transcriptional regulatory elements such as the +5.8-kb site and the ABO promoter appear to decrease transcription from the A- and B-alleles, resulting in reduction in A- and B-antigen expression in A3 and B3, respectively.
    Vox Sanguinis 03/2014; · 2.85 Impact Factor
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    Transfusion 01/2014; · 3.53 Impact Factor
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    01/2014;
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    ABSTRACT: We encountered a broadly reactive red cell alloantibody in 1991, reacting unlike any other known antibody, and named it anti-KANNO after the first patient. A total of 28 cases of anti-KANNO in the Japanese literature were reviewed. To distinguish KANNO from other antibodies against high-frequency antigens, including anti-JMH, anti-Ch/Rg, and anti-Jr(a), we conducted serologic studies with proteolytic enzyme and chemical treatments, complement sensitization against red cells, and serum neutralization techniques. Reactivity of anti-KANNO against red cells lacking high-frequency antigens and antisera to high-frequency antigens against KANNO cells were tested. Among the 28 patients, 26 were female, of whom 25 had a history of pregnancy. Red cells from patient KANNO were reactive with antisera against antigens of high frequency. Anti-KANNO reacted weakly with all cells known to lack high-frequency antigens. It reacted with 2-aminoethylisothiouronium bromide, so it can be distinguished from anti-JMH. Differences among anti-KANNO, anti-Ch/Rg, and anti-Jr(a) emerged with enzyme-treated cells, complement-sensitized cells, and the addition of normal serum. As yet, there are no reports of hemolytic transfusion reaction or hemolytic disease of the fetus and newborn attributable to anti-KANNO. It appears that anti-KANNO is a newly characterized antibody more likely stimulated by pregnancy than by transfusion and with little or no clinical significance. Further surveillance and investigation of anti-KANNO, its antigen biochemistry, and its genetics are warranted.
    Transfusion medicine reviews 12/2013; · 3.61 Impact Factor
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    ABSTRACT: The Kidd blood group system consists of three common phenotypes: Jk(a+b−), Jk(a−b+) and Jk(a+b+), and one rare phenotype, Jk(a−b−). Jka/Jkb polymorphism is associated with c.838G>A (p.Asp280Asn) in exon 9 of the JK (SLC14A1) gene, and the corresponding alleles are named JK*01 and JK*02. The rare phenotype Jk(a−b−) was first found in a Filipina of Spanish and Chinese ancestry, and to date, several JK null alleles responsible for the Jk(a−b−) phenotype have been reported. We report seven novel JK null alleles, 4 with a JK*01 background and 3 with a JK*02 background, identified from Jk(a−b−) Japanese.
    Vox Sanguinis 12/2013; · 2.85 Impact Factor
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    ABSTRACT: An erythroid cell-specific regulatory element, referred to as the +5·8-kb site, had been identified in the first intron of the human ABO blood group gene. Subsequent studies revealed that either a 5·8-kb deletion including the +5·8-kb site or disruption of a GATA factor binding motif at the site was present in all Bm and ABm individuals examined. We investigated the molecular mechanism of the Am phenotype, which is analogous to the Bm phenotype. Genomic DNAs were prepared from peripheral blood of two Am individuals, and the nucleotide sequences were investigated using PCR and direct sequencing. Electrophoretic mobility shift assay (EMSA) and promoter assay with K562 cells were carried out. A novel 23-bp nucleotide deletion was found at the +5·8-kb site in both individuals. EMSAs demonstrated binding of the transcription factor RUNX1 to the nucleotides within the deletion. Promoter assays showed that the deletion reduced the transcriptional activity of the +5·8-kb site. Deletion of the 23-bp nucleotides including the RUNX1 binding site decreases transcription of the A allele, resulting in the reduction in A antigen expression in the Am phenotype.
    Vox Sanguinis 09/2013; · 2.85 Impact Factor
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    ABSTRACT: The number of KEL alleles associated with new antigens or loss of expression of high-prevalence antigens continues to increase. We investigated KEL in five samples: two with K0 (null) phenotypes and three with normal Kell expression and antibodies to high-prevalence antigens. Red blood cell (RBC) typing and antibody identification were by standard methods. Genomic DNA was isolated from white blood cells and DNA array testing and sequencing of KEL exons was performed by standard methods. Proband 1, an Asian woman with Kp(b+) RBCs, presented with alloanti-Kp(b) . Four years later, the antibody was reactive with all RBCs except K0 . She was homozygous for KEL c.877C>T change (p.Arg293Trp), and the high-prevalence antigen absent from her RBCs was named KHUL. Probands 2 and 3, both Japanese and homozygous for KEL c.875G>A (p.Arg292Gln), presented with an antibody reactive with all except K0 RBCs. The antibody, named KYOR, recognizes an antigen antithetical to KYO (KEL31). Proband 4, a pregnant Middle Eastern woman, presented with alloanti-Kp(b) , but her RBCs did not express Kell antigens. She was homozygous for KEL c.230G>T (p.Cys77Phe). Proband 5, a multiply transfused Caucasian female with an antibody reactive with all RBCs except K0 and lacking Kell antigens, was a compound heterozygote carrying a silenced allele c.574C>T (p.Arg192Stop) in trans to c.1664G>A (p.Gly555Glu). We describe two new high-prevalence Kell antigens, KHUL (ISBT 006037; KEL37) and KYOR (ISBT 006038; KEL38), and two novel alleles encoding K0 phenotypes. We caution that antibodies produced by individuals with K0 RBCs or lacking high-prevalence antigens can present as anti-Kp(b) .
    Transfusion 08/2013; · 3.53 Impact Factor
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    ABSTRACT: BACKGROUND: The ABO blood group is important in blood transfusion. Recently, an erythroid cell-specific regulatory element has been identified in the first intron of ABO using luciferase reporter assays with K562 cells. The erythroid cell-specific regulatory activity of the element was dependent upon GATA-1 binding. In addition, partial deletion of Intron 1 including the element was observed in genomic DNAs obtained from 111 Bm and ABm individuals, except for one, whereas the deletion was never found among 1005 individuals with the common phenotypes. STUDY DESIGN AND METHODS: In this study, further investigation was performed to reveal the underlying mechanism responsible for reduction of B antigen expression in the exceptional Bm individual. Peptide nucleic acid-clamping polymerase chain reaction was carried out to amplify the B-related allele, followed by sequence determination. Electrophoretic mobility assays and promoter assays were performed to examine whether a nucleotide substitution reduced the binding of a transcription factor and induced loss of function of the element. RESULTS: Sequence determination revealed one point mutation of the GATA motif in the element. The electrophoretic mobility shift assays showed that the mutation abolished the binding of GATA transcription factors, and the promoter assays demonstrated complete loss of enhancer activity of the element. CONCLUSION: These observations suggest that the mutation in the GATA motif of the erythroid-specific regulatory element may diminish the binding of GATA transcription factors and down regulate transcriptional activity of the element on the B allele, leading to reduction of B antigen expression in erythroid lineage cells of the Bm individual.
    Transfusion 04/2013; · 3.53 Impact Factor
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    ABSTRACT: The ABO blood group is of great importance in blood transfusion and organ transplantation. However, the mechanisms regulating human ABO gene expression remain obscure. On the basis of DNase I-hypersensitive sites in and upstream of ABO in K562 cells, in the present study, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays indicated a novel positive regulatory element in intron 1. This element was shown to enhance ABO promoter activity in an erythroid cell-specific manner. Electrophoretic mobility-shift assays demonstrated that it bound to the tissue-restricted transcription factor GATA-1. Mutation of the GATA motifs to abrogate binding of this factor reduced the regulatory activity of the element. Therefore, GATA-1 appears to be involved in the cell-specific activity of the element. Furthermore, we found that a partial deletion in intron 1 involving the element was associated with B(m) phenotypes. Therefore, it is plausible that deletion of the erythroid cell-specific regulatory element could down-regulate transcription in the B(m) allele, leading to reduction of B-antigen expression in cells of erythroid lineage, but not in mucus-secreting cells. These results support the contention that the enhancer-like element in intron 1 of ABO has a significant function in erythroid cells.
    Blood 03/2012; 119(22):5301-10. · 9.78 Impact Factor
  • Japanese Journal of Transfusion and Cell Therapy 01/2011; 57(6):478-483.
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    ABSTRACT: As a national study, we evaluated the frequencies of irregular erythrocyte antibodies (Abs) by gender and history of transfusion or pregnancy. In total, data from 248,785 patients were analyzed, from whom 4222 irregular erythrocyte Abs were detected in 3554 cases (1.43%). Abs frequencies in these 4222 cases were as follows: anti-E, 26%; anti-Le(a), 26%; anti-P(1), 11%; anti-M, 6%; anti-E+c, 4%; anti-Fy(b), 4%; anti-Di(a), 3%; anti-Le(b), 3%; and anti-D, 2%. In pregnancy, anti-D (5%), anti-Jr(a) (3%) and anti-E+c (6%) Abs were, with statistical significance, more frequent. Among transfused patients, anti-E (38%), anti-E+c (8%), anti-Jk(a) (4%), anti-e+C (2%) and anti-E+Jk(a) (1%) Abs were, with statistical significance, more frequent.
    Transfusion and Apheresis Science 08/2010; 43(1):3-8. · 1.23 Impact Factor
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    ABSTRACT: Rh-associated glycoprotein (RhAG) is closely associated with the Rh proteins in the red cell membrane. Two high frequency antigens (Duclos and DSLK) and one low frequency antigen (Ol(a)) have serological characteristics suggestive of expression on RhAG. RHAG was sequenced from the DNA of one Duclos-negative, one DSLK-negative, and two Ol(a+) individuals. Recombinant protein was expressed in HEK 293 cells. Protein models with RhAG subunits were constructed. The original Duclos-negative patient was homozygous for RHAG 316C>G, encoding Gln106Glu. HEK 293 cells expressing Gln106Glu mutant RhAG did not react with anti-Duclos. An individual with DSLK-negative red cells was homozygous for 490A>C, encoding Lys164Gln. Two Ol(a+) members of the original Norwegian family were heterozygous for 680C>T, encoding Ser227Leu. A Japanese donor with Rh(mod) phenotype had Ol(a+) red cells and was homozygous for 680C>T. The three red cell antigens encoded by RHAG form the RHAG blood group system: Duclos is RHAG1 (030001); Ol(a) is RHAG2 (030002); and DSLK is provisionally RHAG3 (030003).
    Vox Sanguinis 09/2009; 98(2):151-9. · 2.85 Impact Factor
  • Vox Sanguinis 01/2009; 97(s1):45. · 2.85 Impact Factor
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    ABSTRACT: Detection of antibodies against human leucocyte antigens (HLA) and human platelet antigens (HPA) is crucial for patients refractory to platelet transfusion therapy. However, a reliable and high-throughput method for HLA cross-matching and detecting HPA antibodies has not yet been described. Immunocomplex capture fluorescence analysis (ICFA) was developed for high-throughput, simultaneous detection of HLA and HPA antibodies. Microarray beads were separately coupled with monoclonal antibodies specific for CD36, CD41, CD42b, CD49b, CD61 and HLA class I antigens. Platelets reacting with patient serum were lysed and the lysates were incubated with the bead mixture to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins or HLA antigens. The beads capturing immunocomplexes were then subjected to flow cytometric analysis. Immunocomplex capture fluorescence analysis was validated using 50 serum samples containing HLA antibodies and 20 serum samples containing HPA antibodies. The method enabled the detection of all the HLA antibodies with a sensitivity comparable to that of the purified HLA antigen-coated pooled-bead assay (FlowPRA, One Lambda, Canoga Park, CA, USA). The method also enabled the detection of all the HPA antibodies with a sensitivity higher than that of the mixed passive haemagglutination. In this study, we developed a rapid, simple and reliable method for the simultaneous analysis of HLA and HPA antibodies. ICFA can also be used as an alternative to the lymphocyte cytotoxicity test for HLA cross-matching.
    Vox Sanguinis 01/2009; 96(3):244-51. · 2.85 Impact Factor

Publication Stats

627 Citations
159.20 Total Impact Points

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Institutions

  • 1994–2014
    • Japanese Red Cross
      Edo, Tōkyō, Japan
  • 2013
    • Gunma University
      Maebashi, Gunma Prefecture, Japan
  • 2000–2001
    • The University of Tokyo
      Tōkyō, Japan
    • Fukushima Medical University
      • Division of Medicine
      Hukusima, Fukushima, Japan