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Introduction: Rapid antigen detection (RAD) tests are convenient tools for detecting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinics, and testing using saliva samples could decrease the risk of infection during sample collection. This study aimed to assess the accuracy of the SARS-CoV-2 RAD for testing of nasopharyngeal swab specimens and saliva samples in comparison with the RT-PCR tests and viral culture for detecting viable virus., Methods: One hundred seventeen nasopharyngeal swab specimens and 73 saliva samples with positive results on RT-PCR were used. Residual samples were assayed using a commercially available RAD test immediately, and its positivity was determined at various time points during the clinical course. The concordance between 54 nasopharyngeal swab samples and saliva samples that were collected simultaneously was determined. Viral culture was performed on 117 samples and compared with the results of the RAD test., Results: The positive rate of RAD test using saliva samples was low throughout the clinical course. Poor concordance was observed between nasopharyngeal swab specimens and saliva samples (75.9%, kappa coefficient 0.310). However, a substantially high concordance between the RAD test and viral culture was observed in both nasopharyngeal swab specimens (86.8%, kappa coefficient 0.680) and saliva samples (95.1%, kappa coefficient 0.643)., Conclusions: The sensitivity of the SARS-CoV-2 RAD test was insufficient, particularly for saliva samples. However, a substantially high concordance with viral culture suggests its potential utility as an auxiliary test for estimating SARS-CoV-2 viability., (Copyright © 2021 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is a rare autoimmune disorder caused by neutralizing anti-ADAMTS13 autoantibodies. In white individuals, HLA allele DRB1*11 is a predisposing factor for iTTP, whereas DRB1*04 is a protective factor. However, the role of HLA in Asians is unclear. In this study, we analyzed 10 HLA loci using next-generation sequencing in 52 Japanese patients with iTTP, and the allele frequency in the iTTP group was compared with that in a Japanese control group. We identified the following HLA alleles as predisposing factors for iTTP in the Japanese population: DRB1*08:03 (odds ratio [OR], 3.06; corrected P [Pc] = .005), DRB3/4/5*blank (OR, 2.3; Pc = .007), DQA1*01:03 (OR, 2.25; Pc = .006), and DQB1*06:01 (OR,: 2.41; Pc = .003). The estimated haplotype consisting of these 4 alleles was significantly more frequent in the iTTP group than in the control group (30.8% vs 6.0%; Pc < .001). DRB1*15:01 and DRB5*01:01 were weak protective factors for iTTP (OR, 0.23; Pc = .076; and OR, 0.23, Pc = .034, respectively). On the other hand, DRB1*11 and DRB1*04 were not associated with iTTP in the Japanese. These findings indicated that predisposing and protective factors for iTTP differ between Japanese and white individuals. HLA-DR molecules encoded by DRB1*08:03 and DRB1*11:01 have different peptide-binding motifs, but interestingly, bound to the shared ADAMTS13 peptide in an in silico prediction model., (© 2020 by The American Society of Hematology.)

Invasive fungal disease (IFD) is an important infectious complication of hematological disorders, especially in hematopoietic stem cell transplantation recipients. Evidences suggest seasonal and/or geographical variations in the airborne fungal counts and a relationship between those counts and the incidence of IFD. We evaluated the concentrations of indoor airborne fungi quantitated over the course of one year in a hematology ward in Japan. In January, April, July, and October, fixed volumes of air samples were obtained by an air sampler in a hematology ward not equipped with a high-efficiency particulate air filter and incubated in fugal cultures. Samples were also obtained from a protective environment in the same ward and were evaluated. The number of fungal colonies per 50 L of sampled air was highest in October (median 2.25 (range, 0.2-7.0)), which was significantly higher than those in the other three months (0.1 (range, 0-1.0) in January; 0 (0-0) in April; 0.55 (0-2.5) in July; P < 0.01)). Commonly identified pathogens included Penicillium and Cladosrporium species, but Aspergillus species was detected only in July and October samples. These results suggest a seasonal variation in indoor airborne fungal concentrations in Japan, which could affect the epidemiology of IFD., Competing Interests: Declaration of Competing Interest Takehiko Mori received research funding from MSD, Novartis Pharma, LSI Medience, Medical & Biological Laboratories, and Asahi Kasei Corporation, and personal fees from Pfizer Inc., MSD, Janssen Pharma, Sumitomo Dainippon Pharma, Novartis Pharma, Kyowa Kirin, Chugai Pharmaceutical, Shionogi & Co., Japan Blood Products Organization, Takeda Pharmaceutical, Ono Pharmaceutical, Shire, Eisai, and Astellas Pharma; Taku Kikuchi received personal fees from Janssen Pharma; Jun Kato received personal fees from Pfizer Inc., Janssen Pharma, and Astellas Pharma; Naoki Hasegawa received personal fees and research funding from Pfizer Inc., MSD, Daiichi Sankyo Co., Ltd, Taisho Pharmaceutical Co., Ltd.,Eisai Co., Ltd, Sumitomo Dainippon Pharma, and Astellas Pharma, and grant from Insmed Incorporated; Shinichiro Okamoto received research funding from Sumitomo Dainippon Pharma, Pfizer, Teijin pharma, Novartis Pharma, Bristol-Myers Squibb, Mochida Pharmaceutical, JCR Pharmaceuticals, Toyama Chemical, Takeda Pharmaceutical, Daiichi Sankyo, Shionogi & Co., Sanofi, Kyowa Kirin, Ono Pharmaceutical, Otsuka Pharmaceutical, Eisai, Asahi Kasei Corporation, and Japan Blood Products Organization, and personal fees from Pfizer Inc., Janssen Pharma, and Astellas Pharma., (Copyright © 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The QuantiFERON TB Gold Plus (QFT-Plus) test is a newly approved interferon-gamma releasing assay test for detecting latent tuberculosis. Although blood samples for QFT test can be refrigerated for 48 h in lithium-heparin tubes according to package inserts, no published data are available on the effects of sample refrigeration on the test results. We conducted a clinical study that aimed to elucidate whether sample refrigeration for 48 h affects QFT-Plus test results. We collected 2 blood samples each from 40 participants for QFT-Plus; one sample was refrigerated before incubation for QFT-Plus assay, while the other sample was incubated soon after collection and treated as control. After comparing QFT-Plus test results of refrigerated samples and control samples, the concordance rate and kappa coefficient between them were 95% and 0.90, respectively. Thus, blood samples for QFT-Plus test can be refrigerated for 48 h in lithium-heparin tubes without influencing the test results., Competing Interests: Declarations of Competing Interest The funder of this study is a QFT-Plus distributor in Japan., (Copyright © 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Mycobacterium avium complex refers to a group of environmental bacteria which inhabit water and soil. Although Mycobacterium avium complex is capable of causing refractory lung infections, the risk factors for Mycobacterium avium complex lung disease are still unclear. This study aimed to determine the associations between Mycobacterium avium complex lung disease and soil or water exposure. Using questionnaires along with clinical data, we compared soil exposure, along with bathtub bathing and showering habits between 172 women with Mycobacterium avium complex lung disease and 339 women without Mycobacterium avium complex infection as controls. Showering was found to be independently associated with the presence of Mycobacterium avium complex lung disease (adjusted odds ratio 5.72, 95%, confidence interval 1.99 to 16.46). Although the mean age of the groups was different, an age-matched sub-analysis yielded similar results. These results indicate that showering may be a risk factor for Mycobacterium avium complex lung disease., (Copyright © 2019 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The clinical need for platelet transfusions is increasing; however, donor-dependent platelet transfusions are associated with practical problems, such as the limited supply and the risk of infection. Thus, we developed a manufacturing system for platelets from a donor-independent cell source: a human adipose-derived mesenchymal stromal/stem cell line (ASCL). The ASCL was obtained using an upside-down culture flask method and satisfied the minimal criteria for defining mesenchymal stem cells (MSCs) by The International Society for Cellular Therapy. The ASCL showed its proliferation capacity for ≥2 months without any abnormal karyotypes. The ASCL was cultured in megakaryocyte induction media. ASCL-derived megakaryocytes were obtained, with a peak at day 8 of culture, and ASCL-derived platelets (ASCL-PLTs) were obtained, with a peak at day 12 of culture. We observed that CD42b + cells expressed an MSC marker (CD90) which is related to cell adhesion. Compared with peripheral platelets, ASCL-PLTs exhibit higher levels of PAC1 binding, P-selectin surface exposure, ristocetin-induced platelet aggregation, and ADP-induced platelet aggregation, as well as similar levels of fibrinogen binding and collagen-induced platelet aggregation. ASCL-PLTs have lower epinephrine-induced platelet aggregation. The pattern of in vivo kinetics after infusion into irradiated immunodeficient NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ mice was similar to that of platelet concentrates. ASCL-PLTs have similar characteristics to those of peripheral platelets and might have an additional function as MSCs. The establishment of the ASCL and its differentiation into ASCL-PLTs do not require gene transfer, and endogenous thrombopoietin is used for differentiation. The present protocol is a simple method that does not require feeder cells, further enhancing the clinical application of our approach., (© 2019 by The American Society of Hematology.)

Background: Long-term follow-up of studies to investigate preventive effects of aspirin on arterial thrombosis indicate that aspirin reduces the incidence and mortality of some cancers in Western populations., Objectives: To explore the effects of aspirin on cancer incidence and mortality in the elderly Japanese., Patients/methods: Patients aged 60 to 85 years, presenting with hypertension, dyslipidemia, or diabetes mellitus (n = 14 601, 7297 in the aspirin group and 7304 in the no-aspirin group) participated the Japanese Primary Prevention Project (JPPP), a multicenter, open-label, randomized, parallel-group trial. A subanalysis of JPPP was performed to analyze the incidence of newly diagnosed cancer and death related to cancer., Results: The cumulative incidence of newly diagnosed cancer was 5.60% (4.65-6.64%) in the aspirin group and 4.14% (3.67-4.66%) in the no-aspirin group. The hazard ratio for newly diagnosed cancer was 1.24 (1.06-1.46), and the cancer incidence was significantly higher in the aspirin group. The cumulative cancer mortality was 1.96% (1.65-2.31%) in the aspirin group and 1.87% (1.56-2.22%) in the no-aspirin group, with no statistically significant difference. The Fine and Gray model suggested that the difference in the incidence of newly diagnosed cancer between the two groups decreased year by year., Conclusions: Low-dose aspirin use did not reduce the cancer incidence or cancer mortality during a 5-year-average study period in the elderly Japanese. The cancer incidence in the aspirin group might decrease, however, to less than that in the no-aspirin group after the study period. Aspirin use might have led to earlier cancer diagnosis in our study.

Combined effects of penicillin (PEN) and gentamicin (GM) against Streptococcus agalactiae, i.e. group B streptococci (GBS), are known to occur, but synergy has not been examined in strains with reduced PEN susceptibility, usually called PEN-resistant GBS (PRGBS). We therefore studied combined effects of β-lactam antibiotics and GM in cultures of 3 PRGBS strains belonging to serotype Ia or III that were isolated from Japanese adults with invasive infections. Killing kinetics were determined at 2-h intervals from 0 to 6 h after exposure to ampicillin (AMP) or cefotaxime (CTX) combined with GM. Concentrations of GM in bacterial cells were measured by liquid chromatography-tandem mass spectrometry. Morphologic changes after exposure to agents were observed by transmission electron microscopy. Combining AMP or CTX with GM synergistically increased bactericidal activity against PRGBS beyond that of either β-lactam alone. GM concentrations in bacterial cells increased 5- to 8-fold when GM was combined with AMP or CTX. Electron microscopically, bacterial cells showed aggregates of strands and ribosomal damage most likely reflecting enhanced GM uptake into bacterial cells. This uptake appeared to result from cell wall damage caused by β-lactam antibiotics. This study suggests that combining β-lactam antibiotics with GM might be useful against severe PRGBS infection., (Copyright © 2017 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: A novel chemiluminescent enzyme immunoassay (CLEIA) was recently developed to quantify autoantibodies specific for desmogleins (Dsgs) and BP180, the target antigens of pemphigus and pemphigoid. This assay is automated and highly accurate and efficient., Objective: To validate the use of the CLEIA for detection of autoantibodies during the clinical courses of patients with pemphigus and pemphigoid., Methods: To define cut-off values for Dsg1, Dsg3, and BP180, we evaluated 47 serum samples from patients with pemphigus foliaceus (PF), 59 from those with pemphigus vulgaris (PV), 52 from those with bullous pemphigoid (BP), and 995 from healthy individuals. We also evaluated any fluctuations in CLEIA titers according to disease activity during the clinical course of 10 cases each of PF, PV, and BP. We used clinical symptom scores, the pemphigus disease area index (PDAI) and the bullous pemphigoid disease area index (BPDAI), to evaluate disease activity., Results: The cut-off values for the CLEIA titers determined by the Youden index were 15.4U/mL for Dsg1, 14.9U/mL for Dsg3, and 16.8U/mL for BP180. CLEIA titers fluctuated in parallel with the PDAI/BPDAI scores in 28 of the 30 cases with PF, PV, or BP. Although the CLEIA and enzyme-linked immunosorbent assay (ELISA) values in the same samples differed substantially in some cases, the concordance rates of positive/negative results between the CLEIA and ELISA were 96% for Dsg1, 97% for Dsg3, and 96% for BP180., Conclusion: The CLEIA, a newly developed, highly effective autoantibody detection system, is as reliable as ELISA for evaluation of the clinical courses of pemphigus and pemphigoid., (Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

The development of an effective rat model of incisional surgical site infection (SSI) has so far proven difficult. In this study, we created a novel incisional SSI model and validated it in terms of both macroscopic and microscopic aspects including its response to treatment using antimicrobial wound-dressing, Aquacel Ag(®). Wounds were created on the dorsum of rats. 3-0 Vicryl(®) threads inoculated with Escherichia coli were inserted in the wound beds in the infection group (n = 6). The wounds were closed for two days to induce infection and then opened and covered with polypropylene sheets during the study. Aquacel Ag was placed under the polypropylene sheet in the infected wounds of the Aquacel Ag group rats (n = 6). The wounds in the control group (n = 6) contained sterile Vicryl thread that had not been inoculated with E. coli. The macroscopic appearance, wound area, bacterial counts, and histology of each group were evaluated. The infection group demonstrated significantly lower wound healing (p < 0.001), greater bacterial counts (median [interquartile range] ratings, 2.15 × 10(7) [0.51 × 10(7)-53.40 × 10(7)] vs 2.07 × 10(4) [0.60 × 10(4)-4.45 × 10(4)] CFU/g, respectively; p < 0.01), and severer histological inflammation (p < 0.001) than the control group. The Aquacel Ag group was only able to show significantly better wound healing than the infection group (p < 0.001). The new incisional SSI model exhibited all clinical manifestations of incisional SSI. It could be utilized to assess the effectiveness of newly developed treatments for incisional SSI., (Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

We describe a 91-year-old woman who suffered from fungal keratitis after corneal transplantation. The causative organism was identified as Wickerhamomyces anomalus (formerly Pichia anomala or Hansenula anomala) on the basis of morphological characteristics and the sequence of the internal transcribed spacer region of the ribosomal RNA gene. The patient was successfully treated with topical micafungin (MCFG) only. We present the first report of a case of W. anomalus fungal keratitis that responded to topical treatment with the antifungal MCFG., (Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Background: In newborn infants, there are no reference intervals for urinary free steroids, which are thought to reflect the bioavailable fraction of steroids in the blood. We establish a method for simultaneous measurement of urinary free adrenal steroids such as pregnenolone, progesterone, 16α-hydroxyprogesterone, 17α-hydroxyprogesterone, 21-deoxycortisone, 21-deoxycortisol, dehydroepiandrosterone, androstenedione, and 11β-hydroxyandrostenedione by using stable isotope dilution gas chromatography/mass spectrometry (SID-GC/MS) and determined the reference intervals for urinary levels of free adrenal steroids in Japanese newborn infants., Methods: Newborn pooled urine was used for validation. Spot urine samples were collected from 67 full-term Japanese newborn infants (34 male and 33 female infants) at 3-4 days of age to determine reference intervals. The extracted and purified free steroids were delivered with heptafluorobutyric anhydride and analyzed by SID-GC/MS., Results: We validated a SID-GC/MS method with good repeatability and recovery rate. The preliminary reference intervals (median [range], μmol/mol creatinine) were as follows: pregnenolone, 4.2 (0.7-31.6); progesterone, 0.5 (not detected (n.d.)-0.6); 16α-hydroxyprogesterone, 1.4 (n.d.-10.3); 17α-hydroxyprogesterone, 1.1 (n.d.-1.9); 21-deoxycortisone, n.d. (n.d.-n.d.); 21-deoxycortisol, n.d. (n.d.-n.d.); dehydroepiandrosterone, 2.2 (0.6-27.3); androstenedione, 0.7 (n.d.-5.2); and 11β-hydroxyandrostenedione, 2.9 (n.d.-26.7)., Conclusions: We established a reliable SID-GC/MS method and were able to determine preliminary reference intervals for 9 urinary free adrenal steroids in newborn infants., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Determinant factors leading from stem cells to megakaryocytes (MKs) and subsequently platelets have yet to be identified. We now report that a combination of nuclear factor erythroid-derived 2 p45 unit (p45NF-E2), Maf G, and Maf K can convert mouse fibroblast 3T3 cells and adult human dermal fibroblasts into MKs. To screen MK-inducing factors, gene expressions were compared between 3T3 cells that do not differentiate into MKs and 3T3-L1 cells known to differentiate into MKs. 3T3 cells transfected with candidate factors were cultured in a defined MK lineage induction medium. Among the tested factors, transfection with p45NF-E2/MafG/MafK lead to the highest frequency of CD41-positive cells. Adult human dermal fibroblasts transfected with these genes were cultured in MK lineage induction medium. Cultured cells had megakaryocytic features, including surface markers, ploidy, and morphology. More than 90% of MK-sized cells expressed CD41, designated induced MK (iMK). Infusion of these iMK cells into immunodeficient mice led to a time-dependent appearance of CD41-positive, platelet-sized particles. Blood samples from iMK-infused into thrombocytopenic immunodeficient mice were perfused on a collagen-coated chip, and human CD41-positive platelets were incorporated into thrombi on the chip, demonstrating their functionality. These findings demonstrate that a combination of p45NF-E2, Maf G, and Maf K is a key determinant of both megakaryopoiesis and thrombopoiesis.

The rapid diagnosis of pathogens and prompt initiation of appropriate antibiotic therapy are critical factors to reduce the morbidity and mortality associated with sepsis. In this study, we evaluated a multiplex polymerase chain reaction (PCR-M) test that detects bacteria and fungi in whole-blood specimens, comparing its features to those of a blood culture (BC). Following evaluation of the performance for sensitivity and specificity of PCR-M, 78 blood samples from 54 patients with suspected bacterial infections were evaluated. Whole-blood samples for PCR-M were collected at the same time as BC, and PCR-M results were compared with BC results. As a result, minimum sensitivity of the kit was 1-100 cfu/ml. The PCR-M test correctly identified specificity for 13 out of 14 strains blinded to the assay analyst. Of 78 blood samples examined, 56 (72%) were negative by both methods, and 22 (28%) were positive by at least one of the two methods. PCR-M detected organisms in 21 cases (27%) compared with 12 cases (15%) in BC. The correlation of positives between PCR-M and BC was 92% (11/12), and both methods identified the same organisms in these 11 cases. With higher positive rate compared with BC, PCR-M could detect and identify potentially significant microorganisms within a few hours by using a small volume of a single whole-blood sample. Early detection of microorganisms has the potential to facilitate early determination of appropriate treatment and antimicrobial selection.

Platelets are produced from megakaryocytes (MKs), although the pathway leading from stem cells to MK lineages are not yet fully understood. Recently, we reported to obtain abundant MKs and platelets from human subcutaneous adipose tissues. Adipose tissues contain various cell types, most of which are lineage cells from mesenchymal or adipocyte-derived stem cells, distinct from hematopoietic cells. To identify the cells responsible for the differentiation MK lineages in adipose tissues, this study examined whether the preadipocyte cell line 3T3-L1 and fibroblast cell line 3T3 differentiated into MK lineages in vitro. Cells were cultured in megakaryocyte lineage induction medium. By day 4, most of 3T3 cell-derived cells leaded to cell death. In contrast, 3T3-L1-derived cells on days 8 showed to have typical characterizations of MK lineages in analyses for specific marker, DNA ploidy, transmission electro micrograph. 3T3-L1-derived platelet-sized cells on day 12 could be stimulated by ADP and PAR4-activating peptide. This study clearly shows in vitro differentiation from 3T3-L1 cells, not from 3T3 cells, into MK lineages., (Copyright © 2010 Elsevier Inc. All rights reserved.)

ADAMTS13 cleaves multimeric von Willebrand factor (VWF) to regulate VWF-mediated thrombus formation. To search ADAMTS13 peptide sequences binding to VWF, a lambda-phage library expressing various peptides of ADAMTS13 on the surface was screened using VWF either immobilized or in solution under static condition. By the first screening, peptides sharing the C-terminus of spacer domain from Arg(670) to Gln(684) (epitope-A) were selected. To explore additional sites, peptide sequences from the first screening were synthesized and added to the second screening. Consequently, Pro(618) to Glu(641) (epitope-B) in the middle of spacer domain was obtained from immobilized VWF condition. Synthetic epitope-B peptide inhibited the cleavage of VWF by ADAMTS13, while the synthetic epitope-A peptide did not as efficiently as epitope-B. Elimination of four amino acids from either sides of epitope-B terminus markedly reduced the inhibitory effect. These two sites in the spacer domain may play significant roles in binding to VWF., (Copyright 2009 Elsevier Inc. All rights reserved.)

Recent advances in regenerative medicine have created a broad spectrum of stem cell research. Among them, tissue stem cell regulations are important issues to clarify the molecular mechanism of differentiation. Adipose tissues have been shown to contain abundant preadipocytes, which are multipotent to differentiate into cells including adipocytes, chondrocytes, and osteoblasts. In this study, we have first shown that megakaryocytes and platelets can be generated from adipocyte precursor cells. Human adipocyte precursor cells were cultured in conditioned media for 12 days to differentiate adipocytes, followed by 12 days of culture in media containing thrombopoietin. The ultrastructures of adipocyte precursor cell- and bone marrow CD34-positive cell-derived megakaryocytes and platelets were similar. In addition, adipocyte precursor cell-derived platelets exhibited surface expression of P-selectin and bound fibrinogen upon stimulation with platelet agonists, suggesting that these platelets were functional. This is the first demonstration that human subcutaneous adipocyte precursor cells can generate megakaryocyte and functional platelets in an in vitro culture system.

Genetic variation, a -1327T/C polymorphism, of human telomerase reverse transcriptase (hTERT) is associated with leukocyte telomere length in healthy subjects, but clinical significances of this functional polymorphism are not clear. Recently, the relationship between the telomere system and coronary artery disease (CAD) was reported. We investigated the association between the -1327T/C polymorphism and (a) susceptibility to CAD and (b) telomere length in CAD patients. In a case-control study, 104 patients confirmed by coronary angiography and 115 age- and sex-matched controls were enrolled. There was a higher frequency of the -1327C/C genotype in CAD patients (51.9%) compared with controls (36.5%, p = 0.0218). Among the 104 CAD patients, leukocyte telomere length in the -1327C/C genotype (7.62+/-2.19 kb, mean+/-SD) was shorter than that in the -1327T/C and -1327T/T genotypes (8.74+/-2.92, p = 0.0287). These findings suggest that the -1327C/C genotype is a genetic risk factor for CAD and relates to shorter telomere length among CAD patients.

alpha2A adrenergic receptor (ADRA2A) on platelets interacts with epinephrine, which has a key role in regulating platelet functions. There is familial clustering of inter-individual variations in the epinephrine-induced platelet aggregation, the molecular basis of which, however, has not been fully understood. In this study, we screened the sequence variations in the transcriptional region of ADRA2A gene and analyzed the relationship between the two common polymorphisms and platelet function using epinephrine/collagen cartridge in the platelet function analyzer-100 system, in a healthy Japanese male population (n=211). Among the identified 16 sequence variations including five novel variations, 1780GG genotype was associated with longer closure time which represents low platelet function under high shear-stress conditions (p=0.0478). We also observed enhanced effect of the combination of 1780GG and 2372AA genotypes on longer closure time (p=0.0319). These findings suggest that 1780A/G and 2372A/G polymorphisms are associated with platelet function in interactions with collagen/epinephrine.

Transcriptional regulation of human telomerase reverse transcriptase (hTERT), a catalytic subunit of telomerase, is essential for telomerase activity associated with telomere length. In this study, we investigated the effects of a (-1327)T/C polymorphism within the hTERT promoter region on the hTERT promoter activity and leukocyte telomere length in normal individuals. The promoter activity in the (-1327)T-sequence was significantly higher than that in the (-1327)C-sequence (p = 0.0004). For leukocyte telomere length, the (-1327)T-allele carriers had significantly longer than the (-1327)T-allele non-carriers (p = 0.0007). Also, there was no age-related shortening in leukocyte telomere length in the (-1327)T/T (p = 0.6633) and (-1327)T/C subjects (p = 0.1691), whereas there was clear age-related telomere shortening in the (-1327)C/C subjects (p = 0.0117). These findings suggest that the functional (-1327)T/C polymorphism of hTERT is associated with leukocyte telomere length in normal individuals.

Congenital thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS) is associated with an inherited von Willebrand factor-cleaving protease (ADAMTS13 [a disintegrin and metalloprotease with thrombospondin type I domains 13]) deficiency. In this study, we identified novel mutations in the ADAMTS13 gene in a patient with TTP. The patient was a 51-year-old Japanese male who exhibited TTP symptoms at frequent intervals. The ADAMTS13 activity during acute episodes was less than 3% that of normal. The enzyme activities of the patient's father and mother were both 46%, and both parents were asymptomatic. Genetic analysis revealed that the patient was a compound heterozygote for 2 mutations. One mutation was a missense mutation in the metalloprotease domain (A250V, exon 7), and the other was a guanine to adenine substitution at the 5' end of intron 3 (intron 3 G-->A). In vitro expression studies revealed that the A250V mutation markedly reduced ADAMTS13 activity and the intron 3 G-->A mutation caused abnormal mRNA synthesis.

The hemostatic activity of von Willebrand factor (vWF) is strongly dependent on its multimeric structure, with the highest activity in 'unusually large' multimers secreted from endothelial cells. The multimeric structure is regulated by a plasma protease, vWF-cleaving protease (vWF-CP, or ADAMTS-13). ADAMTS-13 mRNA is variably expressed in liver and other tissues. Because 15-25% of circulating vWF is stored in platelets, the presence and function of ADAMTS-13 in platelets are important issues. Here we report ADAMTS-13 expression in human platelets. Western blot analysis and flow cytometric analysis on permeabilized platelets revealed the presence of ADAMTS-13 protein. Real-time PCR demonstrated that ADAMTS-13 mRNA is present in platelets of six healthy volunteers, with little quantitative difference. The presence of ADAMTS-13 in platelets may imply the functional role of this enzyme in the local regulation of platelet function at the site of vascular injury or thrombus formation, and provide a useful tool for the analysis of structure and function of this enzyme.

Liposomes carrying both recombinant glycoprotein Ia/IIa (rGPIa/IIa) and Ib alpha (rGPIb alpha) (rGPIa/IIa-Ib alpha-liposomes) instantaneously and irreversibly adhered to the collagen surface in the presence of soluble von Willebrand factor (VWF) at high shear rates, in marked contrast with translocation of liposomes carrying rGPIb alpha alone on the VWF surface. In the absence of soluble VWF, the adhesion of rGPIa/IIa-Ib alpha-liposomes to the collagen surface decreased with increasing shear rates, similar to liposomes carrying rGPIa/IIa alone. While adhesion of liposomes with exofacial rGPIa/IIa and rGPIb alpha densities of 2.17 x 10(3) and 1.00 x 10(4) molecules per particle, respectively, was efficient at high shear rates, reduction in rGPIb alpha density to 5.27 x 10(3) molecules per particle resulted in decreased adhesion even in the presence of soluble VWF. A 50% reduction in the exofacial rGPIa/IIa density resulted in a marked decrease in the adhesive ability of the liposomes at all shear rates tested. The inhibitory effect of antibody against GPIb alpha (GUR83-35) on liposome adhesion was greater at higher shear rates. Further, the anti-GPIa antibody (Gi9) inhibited liposome adhesion more than GUR83-35 at all shear rates tested. These results suggest that the rGPIa/IIa-collagen interaction dominates the adhesion of rGPIa/IIa-Ib alpha-liposomes to the collagen surface at low shear rates, while the rGPIa/IIa-collagen and rGPIb alpha-VWF interaction complements each other, and they synergistically provide the needed functional integration required for liposome adhesion at high shear rates. This study thus has confirmed for the first time the proposed mechanisms of platelet adhesion to the collagen surface under flow conditions using the liposome system.