Yuko Mishima

The frequencies of human neutrophil alloantigens among the Japanese population

Human neutrophil antigens (HNAs) play an important role in a variety of clinical conditions including immune-mediated neutropenia, non-hemolytic transfusion reactions, and transfusion-related acute lung injury. The aim of this study was to investigate the frequency distribution of HNAs-1 to -5 among the Japanese population. We analyzed samples from 570 healthy Japanese by molecular and serologic techniques to estimate the gene frequencies of HNAs-1 to -5. DNA samples were obtained and typed for the HNA-1 (n = 523), -3 (n = 570), -4 (n = 570), and -5 (n = 508), by molecular techniques. The HNA-1 genotype was determined by using a commercial polymerase chain reaction-reverse sequence-specific oligonucleotide probes (PCR-rSSOP) kit. The HNA-3 to -5 genotypes were determined by the PCR-sequence specific primer (PCR-SSP), previously described, with a small modification. The HNA-2a phenotype was determined in 301 donors by granulocyte immunofluorescence test. In Japanese, the gene frequencies of HNA-1a, -1b, and -1c were 0.623, 0.377, and 0.000, respectively. The frequency of HNA-2a phenotype was 0.987, and the gene frequencies of HNA-3a and -3b were 0.654 and 0.346, respectively. HNA-4a and -4b were found at 1.000 and 0.000, respectively, and HNA-5a and -5b at 0.840 and 0.160, respectively. We describe, for the first time, the frequencies of all HNAs (HNA-1 to -5) among the Japanese population. This study will be helpful for the prediction of the risk of alloimmunization to HNA, especially to determine the risk of HNA alloantibody production by transfusion of HNA incompatible blood and feto-maternal incompatibility.

Analytical evaluation of plasma serotonin and sphingosine 1-phosphate and their clinical assessment in early atherosclerosis.

ObjectivesSerotonin stored in platelets is released into plasma on aggregation and activation in atherosclerotic diseases. Sphingosine 1-phosphate (S1P) in plasma is mainly derived from red blood cells and is responsible for the production of nitric oxide in endothelial cells and protects vasculature. The purpose of this study was to investigate the plasma levels of serotonin, S1P, and their clinical relationships with vascular endothelial function in patients with early atherosclerosis. MethodsBlood was withdrawn from patients with low-to-moderate risks of atherosclerotic diseases (n=49, 39±7 years). Platelet-poor plasma was immediately centrifuged. Serotonin levels in plasma were measured with high-performance liquid chromatography. S1P levels in plasma were measured by high-performance liquid chromatography after fluorescent derivatization with o-phthaldialdehyde. Endothelial function was assessed by endothelium-dependent flow-mediated dilation (FMD) and endothelium-independent dilation was measured by glycerol trinitrate-induced dilation using an ultrasound system. ResultsPlasma serotonin was inversely correlated with the FMD value (r=−0.287, P<0.05). Fourteen patients with dyslipidemia, who had not shown improvements after lifestyle modifications, were subsequently treated with rosuvastatin (2.5 mg/day). After 4 weeks of treatment, rosuvastatin improved lipid profiles. Rosuvastatin increased FMD, whereas glycerol trinitrate-induced dilation was unchanged. Notably, percentage decrease in plasma serotonin was inversely correlated with percentage increase in plasma S1P (r=−0.557, P<0.05). ConclusionPlasma serotonin was inversely correlated with FMD and a decrease in plasma serotonin was inversely correlated with an increase in plasma S1P after statin treatment. The results suggested that plasma levels of serotonin and S1P may be useful for the assessment of endothelial function of patients with low-to-moderate risks of atherosclerotic diseases.

Effects of universal vs bedside leukoreductions on the alloimmunization to platelets and the platelet transfusion refractoriness

BACKGROUND Multiple platelet exposure induces anti-HLA and/or anti-HPA antibody production, which may cause platelet transfusion refractoriness (PTR). In Japan, the universal pre-storage leukocyte reduction (ULR) was fully implemented since 2006, but prior to ULR, in our institution, leukocyte reduction filters were routinely used at the bedside (bedside leukoreduction, BSLR) for all onco-hematological patients receiving multiple platelet transfusions. OBJECTIVE We retrospectively compared patients receiving platelet transfusions in the era of ULR with those of BSLR era. MATERIALS AND METHODS Patients of the BSLR group (409 cases) and the ULR group (586 cases) were compared in terms of alloimmunization and immunological PTR. The clinico-pathological features, including gender, history of pregnancy, number of exposed transfusion donors, periods of transfusion, and prior stem cell transplantation were compared, and the risk factors of alloimmunization were determined. RESULTS The antibody detection rate was significantly higher in the ULR compared to BSLR group (8.7% vs. 5.4%), as well as the immunological PTR rate (7.3% vs. 3.2%). By the multivariate analysis, female gender and the number of platelet donor exposure, but not universal leukoreduction or transfusion period, were found to be the risk factors strongly associated with alloantibody formation. CONCLUSION Although ULR may be superior to BSLR in terms of preventing non-hemolytic transfusion reactions, BSLR was found to be as effective as ULR in terms of preventing platelet alloimmunization and refractoriness. Thus, BSLR should be actively indicated as a realistic alternative in developing countries, before the universal leukoreduction is fully implemented.

TNF-α production in NKT cell hybridoma is regulated by sphingosine-1-phosphate: implications for inflammation in atherosclerosis.

ObjectivesNatural killer T (NKT) cells are unique T lymphocytes that recognize glycolipid antigen and produce various cytokines. NKT cells accelerate atherosclerosis in mice. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid and regulates T-lymphocyte trafficking. We aimed to determine the effects of S1P on the production of proinflammatory cytokine, tumor necrosis factor (TNF)-&agr;, in NKT cell hybridomas and mouse NKT cells. Materials and methodsNKT cell hybridomas and sorted mouse NKT cells were stimulated with S1P and &agr;-galactosylceramide (&agr;-GalCer), the major ligand to produce cytokines in NKT cells. TNF-&agr; mRNA expression and protein production were determined by real-time PCR and ELISA, respectively. Cell migration was assayed using chemotaxicell. Plasma S1P was measured using HPLC. ResultsHybridomas expressed S1P receptors, S1P1, S1P2, and S1P4. S1P and &agr;-GalCer increased TNF-&agr; mRNA expression and protein production. S1P enhanced TNF-&agr; induction by &agr;-GalCer. S1P receptor antagonists decreased the TNF-&agr; mRNA expression induced by S1P. FTY720, an immunosuppressive S1P receptor modulator, also decreased the TNF-&agr; mRNA expression. The migration of NKT cell hybridomas was increased by S1P. FTY720 reduced the migration induced by S1P. S1P also increased the TNF-&agr; mRNA expression in mouse NKT cells. Plasma TNF-&agr; levels in patients with high plasma S1P (≥500 nmol/l) were higher than those in patients with low S1P (<500 nmol/l). ConclusionS1P binds to S1P receptors in NKT cells and enhances TNF-&agr; production. TNF-&agr; overproduction may induce atherogenic inflammatory responses. S1P may serve as a novel therapeutic target for amelioration of vascular inflammatory diseases.

The role of alloantibodies against human platelet antigen-15 in multiply platelet transfused patients

Several studies have documented the role of antibodies against human platelet (PLT) antigen (HPA)‐15 in alloimmune‐mediated thrombocytopenia including neonatal alloimmune thrombocytopenia, PLT transfusion refractoriness (PTR), and posttransfusion purpura in Caucasian persons. However, the relevance of anti‐HPA‐15 in PTR among the Japanese population is still unclear.

Possible involvement of sphingomyelin in the regulation of the plasma sphingosine 1-phosphate level in human subjects.

OBJECTIVES Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid mediator. Although the plasma S1P concentration is reportedly determined by cellular components, including erythrocytes, platelets, and vascular endothelial cells, the possible involvement of other factors, such as serum sphingomyelin (SM) and autotaxin (ATX), remains to be elucidated. DESIGN AND METHODS We measured S1P using high-performance liquid chromatography (HPLC), SM and lysophosphatidic acid (LPA) using enzymatic assays, ATX antigen using a two-site enzyme immunoassay, and ATX activity using a lysophospholipase D activity assay. To fractionate the lipoproteins, plasma samples were separated using fast protein liquid chromatography (FPLC) utilizing a Superose 6 column. RESULTS The plasma S1P level was positively correlated with the levels of SM and lysophosphatidylcholine, but not with the level of phosphatidylcholine. Although SM was present in the very low-density lipoprotein (VLDL) fraction, neither the plasma S1P level nor the SM level was affected by feeding. The plasma S1P level was negatively correlated with the ATX activity. Although the incubation of 100 μmol/L of sphingosylphosphorylcholine (SPC) with the serum resulted in a significant increase in the S1P level because of the presence of ATX, the physiological concentration of SPC did not mimic this effect. CONCLUSION The plasma S1P level was affected by the serum SM level, while the possibility of ATX involvement in the increase in the plasma S1P level was considered to be remote at least in healthy human subjects.

The frequencies of SLC44A2 alleles among the Japanese population

The human neutrophil antigens (HNAs) are assigned to five antigen groups, namely HNA-1 to -5 (1). HNA antibodies are implicated in immune neutropenia and transfusion reactions, such as neonatal alloimmune neutropenia, refractoriness to granulocyte transfusion, febrile transfusion reactions, and transfusion-related acute lung injury (TRALI) (1). TRALI is one of the leading causes of transfusion-related morbidity and mortality. HNA antibodies are among the various factors implicated in TRALI induction, and especially HNA-3a alloantibodies have been found in severe cases requiring artificial ventilation, as well as in fatal cases (2). HNA-3 results from a single nucleotide polymorphism (SNP) (A461G) in the choline transporter-like protein (CTL) 2 gene (SLC44A2). The allele SLC44A2*1 , expressing HNA-3a, and SLC44A2*2 , expressing HNA-3b, are due to 461G and the 461A, respectively (3, 4). Furthermore, an additional SNP was described (C457T) (Figure S1; 5–7), which creates a SLC44A2*1 variant (SLC44A2*1:2 ). Recently, we reported the frequencies of HNA-1 to -5 among the Japanese population, and observed a higher frequency of HNA-3b/b in Japanese than in other populations (HNA-3b: 0.346 versus 0.207–0.262) (8). This observation suggests the higher risk of alloimmunization in HNA-3b/b homozygous individuals by exposure to the HNA-3a antigen, through incompatible transfusion and pregnancy, in Japan than in other countries. Thus, the precise detection of HNA-3a antibody is essential for the diagnosis and the treatment of TRALI. Here, the frequency of SLC44A2 alleles, not published in our previous report (8), was investigated in 507 unrelated