Molecular and serological investigation of Francisella tularensis among wild animals in Yamaguchi prefecture.

Francisella tularensis is an intracellular gram-negative bacterium known as the causative agent of tularemia, which can be transmitted to humans by direct contact with wild animals or by tick bites. Although F. tularensis is highly pathogenic, its recent prevalence in Japan is underreported due to the small number of reported cases. To clarify the current situation of F. tularensis in wild animals, we conducted surveillance on various species of wild animals in Yamaguchi prefecture. In this study, we screened 809 samples collected from 90 Japanese black bears, 105 Japanese monkeys, 168 sika deer, 205 wild boars, and 84 bats. For seroprevalence analysis, we tested 177 serum samples from 75 black bears and 102 monkeys using the microagglutination test. The results showed that serums from five black bears exhibited slight agglutination. Western blot was performed as a confirmatory test on these five samples, but no positive signals were detected. Additionally, molecular surveillance was conducted using DNA extracted from 464 whole blood and 168 tissues, targeting the gene encoding 23 KDa hypothetical protein by real-time PCR and outer membrane protein A gene by conventional PCR. No positive samples of F. tularensis were detected by either real-time or conventional PCR. Although we did not detect any F. tularensis-positive samples through serological and molecular analyses, continuous surveillance studies are necessary since sporadic human cases have been reported in Japan.

Inhibitory Effects of Aspirin and Cilostazol on Intracellular Ca2+ Mobilization and Aggregation in Thrombin-activated Human Platelets.

Platelets play an important role in physiological hemostatic mechanisms. In contrast, platelet activation has been implicated in pathological conditions, such as atherosclerosis, angiogenesis, and inflammation. Thrombin is considered to be of particular pathological importance as a platelet-activating substance, and thrombin-activated platelets are detected in the blood of patients with advanced occlusive arterial disease. Ca2+ acts as a second messenger in platelet activation, and the regulation of intracellular Ca2+ concentrations ([Ca2+]i) is important for controlling platelet functions. However, changes in [Ca2+]i by antiplatelet agents remain unclear. Therefore, we herein investigated the relationship between [Ca2+]i and the intensity of platelet aggregation after a thrombin stimulation, the relationship between [Ca2+]i and the intensity of platelet aggregation by antiplatelet agents, and the effects of antiplatelet agents on thrombin-activated platelets as a surrogate platelet model for arterial occlusive disease. Fura2-loaded platelets were treated with phosphate-buffered saline or a low concentration of thrombin (0.005 U/mL), followed by antiplatelet agents (aspirin or cilostazol), and changes in [Ca2+]i and the intensity of platelet aggregation by the thrombin stimulation were measured using fluorescence spectrophotometry. Changes in [Ca2+]i and the intensity of platelet aggregation after the thrombin stimulation as well as the relationship between [Ca2+]i and the intensity of platelet aggregation by antiplatelet agents indicated that cilostazol exerted stronger antiplatelet effects than aspirin and also that antiplatelet effects may be attenuated in thrombin-activated platelets. The present results also suggest the utility of thrombin-activated platelets as a surrogate platelet model for arterial occlusive disease. These results may contribute to future drug development for antiplatelet therapy. J. Med. Invest. 70 : 94-100, February, 2023.

Genetic Variation in Y-Chromosome Genes of Sika Deer (Cervus nippon) in Japan.

Sika deer (Cervus nippon) in Japan are classified into southern and northern groups. However, previous studies primarily relied on maternally inherited mitochondrial DNA (mtDNA). The paternally inherited Y-chromosome is useful for analyzing the contribution of males to the population genetic history of sika deer. In total, approximately 16 kb of partial sequences of four Y-chromosomal genes, Y-linked, sex-determining region Y, DEAD-box helicase 3 Y-linked, and Zinc finger protein Y-linked, were sequenced to investigate intraspecific variation. As a result, we identified nine intronic single nucleotide polymorphisms (SNPs) in 478 sika deer samples collected over the entire Japanese archipelago from Hokkaido to Kyushu. SNP genotyping revealed 10 distinct haplotypes (SYH1-SYH10). The most common haplotype (SYH1) was present in all populations and was the most abundant haplotype, identified in 80.3% of the sampled individuals. The remaining haplotypes were unique to a single locality. SYH1 was also central to all other haplotypes that diverged by a SNP, resulting in this haplotype being the core of a star-like cluster topography. We found that contrary to mtDNA patterns, there was no clear differentiation of Y-chromosome markers between the southern and the northern populations. Due to the female philopatry of sika deer, mtDNA may provide a highly structured differentiation of populations. On the other hand, the male-biased gene flow may provide a reduced differentiation of populations. Our findings revealed that the genetic structure of the Japanese sika deer is more complex than previously thought based on mtDNA-based phylogeographic studies.

Safety and effectiveness of granulocyte and monocyte adsorptive apheresis in patients with inflammatory bowel disease in special situations: a multicentre cohort study.

BACKGROUND: The available information on granulocyte and monocyte adsorptive apheresis (GMA) in patients with inflammatory bowel disease (IBD) under special situations remains unclear. We conducted a retrospective, multicentre cohort study to evaluate the safety and effectiveness of GMA in patients with IBD under special situations. METHODS: This study included patients with ulcerative colitis (UC) or Crohn's disease who had at least one special situation feature and who had received GMA between November 2013 and March 2017. The incidence of adverse events (AEs) was compared in relation to the special situation, and patient background factors related to an AE were identified. For patients with UC, clinical remission was defined as a partial Mayo score of ≤2. RESULTS: A total of 437 patients were included in this study. The incidence of AEs among the elderly patients (11.2%) was similar in all patients (11.4%), whereas the incidences of AEs in patients on multiple immunosuppressant medications (15.2%), patients with anaemia (18.1%) and paediatric/adolescent patients (18.9%) were higher than that in all patients (11.4%). In multivariate analysis, anaemia and concomitant immunosuppressant medications were independently associated with the incidence of AEs. Clinical remission was achieved in 46.4% of the patients with UC. CONCLUSIONS: The incidence of AEs in the elderly patients was not higher than that in all patients, whereas the incidence of AE was higher in patients with anaemia and those on multiple immunosuppressant medications than that in all patients. GMA is a safe treatment option in elderly patients with IBD.

ABO genotyping of various hematopoietic cell lines to select model cells for research purposes.

Various cells from humans and animals have been established as cell lines, and their features, characteristics, and origins have been reported. Many laboratories use cell lines as model cells, which are selected to suit research purposes. We attempted to identify the ABO genotypes of 31 human leukemia and lymphoma cell lines stored in our laboratory using three methods: the PCR amplification of specific alleles (PASA), PCR-restriction fragment length polymorphism (RFLP), and the direct DNA sequencing of PCR products. We distinguished 31 human leukemia and lymphoma cell lines examined into six major ABO genotypes: A/O (A101/O01: n = 1, A101/O12: n = 4, A101/O26: n = 1, A101/O49: n = 1, A102/O01: n = 3), A/A (A101/A101: n = 1, A102/A102: n = 2), B/O (Bw29/O01: n = 1), B/B (B101/B101: n = 2), O/O (O01/O01: n = 9, O01/O02: n = 1, O01/O26: n = 1, O02/O03: n = 1), and A/B (A102/B101: n = 3). To the best of our knowledge, this is the first study to identify the ABO genotypes of various cell lines. The ABO genotypes of cell lines are important when selecting an experimental model cell for an ABO blood group study, and are essential information for cell lines. These results may be employed by research and clinical laboratories as well as in the forensic field.

Interspecific comparison of allometry between body weight and chest girth in domestic bovids.

The sizes of body parts often co-vary through exponential scaling, known as allometry. The evolution of allometry is central to the generation of morphological diversity. To make inferences regarding the evolved responses in allometry to natural and artificial selection, we compared allometric parameters (slope and intercept) among seven species and breeds of domestic bovids using cross-sectional ontogenetic data and attempted to interpret the differences in these parameters. The allometric slopes were not different among some species, whereas those between breeds within species were, indicating that the slopes were typically invariant but could be changed under strong, specific selection. With the exception of yak, the differences in the intercept independent of the slopes (the alternative intercept) among species might better correspond to their divergence times than the differences in allometric slope, and the remarkably higher alternative intercept found in yaks can be explained by their unique morphological evolution. These findings provide evidence that differences in the alternative intercept can retain traces of the phylogenetic changes derived from differentiation and evolution.

Evaluation of effects of various drugs on platelet functions using phorbol 12-myristate 13-acetate-induced megakaryocytic human erythroid leukemia cells.

BACKGROUND: The hyperfunction and activation of platelets have been strongly implicated in the development and recurrence of arterial occlusive disease, and various antiplatelet drugs are used to treat and prevent such diseases. New antiplatelet drugs and many other drugs have been developed, but some drugs may have adverse effects on platelet functions. OBJECTIVE: The aim of this study was to establish an evaluation method for evaluating the effect and adverse effect of various drugs on platelet functions. MATERIALS AND METHODS: Human erythroid leukemia (HEL) cells were used after megakaryocytic differentiation with phorbol 12-myristate 13-acetate as an alternative to platelets. Drugs were evaluated by changes in intracellular Ca2+ concentration ([Ca2+]i) mobilization in Fura2-loaded phorbol 12-myristate 13-acetate-induced HEL cells. Aspirin and cilostazol were selected as antiplatelet drugs and ibuprofen and sodium valproate as other drugs. RESULTS: There was a positive correlation between [Ca2+]i and platelet aggregation induced by thrombin. Aspirin (5.6-560 µM) and cilostazol (5-10 µM) significantly inhibited thrombin-induced increases in [Ca2+]i in a concentration-dependent manner. On the other hand, ibuprofen (8-200 µM) and sodium valproate (50-1,000 µg/mL) also significantly inhibited thrombin-induced increases in [Ca2+]i in a concentration-dependent manner. Furthermore, the interaction effects of the simultaneous combined use of aspirin and ibuprofen or sodium valproate were evaluated. When the inhibitory effect of aspirin was higher than that of ibuprofen, the effect of aspirin was reduced, whereas when the inhibitory effect of aspirin was lower than that of ibuprofen, the effect of ibuprofen was reduced. The combination of aspirin and sodium valproate synergistically inhibited thrombin-induced [Ca2+]i. CONCLUSION: It is possible to induce HEL cells to differentiate into megakaryocytes, which are a useful model for the study of platelet functions, and the quantification of the inhibition of thrombin-induced increases in [Ca2+]i is applicable to the evaluation of the effects of various drugs on platelets.

Flow Cytometric Evaluation of Surface CD56 Expression on Activated Natural Killer Cells as Functional Marker.

Surface CD56 is the most important cell marker for defining NK cells. However, the relationship between the expression of surface CD56 and NK cell activity has not yet been elucidated in detail. Thirteen healthy volunteers were enrolled in the present study. Peripheral blood mononuclear cells (PBMCs) were stimulated with rIL-2 or rIL-12 (1, 10, 100 U/mL) for 18 h at 37°C. After incubation, surface CD56 expression on NK cells was evaluated using a flow cytometric analysis. A colorimetric-based lactate dehydrogenase (LDH) assay was used for experiments on cytotoxicity. IFN-γ mRNA gene expression was quantified by real-time PCR. The expression level of surface CD56 on NK cells, cytotoxicity, and IFN-γ mRNA gene expression were significantly increased by the rIL-2 and rIL-12 stimulations. In addition, a positive correlation was found between surface CD56 expression and cytotoxic activity or IFN-γ mRNA gene expression. We revealed that the quantification of surface CD56 expression was applicable to the evaluation of cytotoxicity and IFN-γ production in activated NK cells. These results suggest that the measurement of surface CD56 expression represent an easy and rapidly reproducible technique to evaluate the activated state of NK cells and monitor NK cell activity in immunotherapy. J. Med. Invest. 63: 199-203, August, 2016.

SNPs rs4656317 and rs12071048 located within an enhancer in FCGR3A are in strong linkage disequilibrium with rs396991 and influence NK cell-mediated ADCC by transcriptional regulation.

CD16 receptors are mainly expresses on the surface of NK cells and mediate antibody-dependent cellular cytotoxicity (ADCC). The authors previously reported that NK cell-mediated ADCC is influenced by the single nucleotide polymorphism (SNP) rs396991 (T>G; F158V), and the structure and expression levels of CD16 differed among these genotypes. The authors examined haplotype frequency distributions among rs396991 and other SNPs, rs10917571 (G>T), rs4656317 (C>G), and rs12071048 (G>A), located in an enhancer of the FCGR3A gene. A total of 101 healthy Japanese were genotyped for the presence of these SNPs. The authors also measured ADCC activity, FCGR3A transcript levels, and surface CD16 expression on NK cells. We found that the regulatory SNPs (rSNPs) rs4656317 and rs12071048 were in strong linkage disequilibrium with rs396991. These two SNPs with major alleles had higher ADCC activity than those with minor alleles. In addition, FCGR3A transcript levels and surface CD16 expression levels were regulated by these SNPs. These findings suggest that NK cell-mediated ADCC could be influenced by transcriptional regulation of these rSNPs. These findings help to clarify our understanding of the linkage disequilibrium among functional SNPs in the FCGR3A gene, and provide a resource for investigating the roles of functional SNPs in NK cell-mediated ADCC.

The influence of NK cell-mediated ADCC: Structure and expression of the CD16 molecule differ among FcγRIIIa-V158F genotypes in healthy Japanese subjects.

NK cells express the CD16 (FcγRIIIa) receptor, which mediates antibody-dependent cellular cytotoxicity (ADCC), on their cell surface. Therefore, ADCC activity may be influenced by qualitative or quantitative changes in the CD16 molecule on NK cells. Responses to NK cell-mediated ADCC have been shown to depend on single nucleotide polymorphisms (SNPs) at FcγRIIIa amino acid position 158. However, a consensus has not yet been reached regarding differences in the structure and expression levels of the CD16 molecule among FcγRIIIa-V158F genotypes, which have not yet been adequately investigated in healthy Japanese individuals. We herein examined the influence of the FcγRIIIa polymorphism on ADCC, binding affinity of CD16 to the Fc region, FCGR3A gene expression, and cell-surface CD16 expression in healthy Japanese subjects. FcγRIIIa-V158F genotyping was performed for 101 subjects. The results obtained showed that all parameters analyzed increased in the order of V/V>V/F>F/F and were significantly higher in V/V subjects than in F/F subjects. Moreover, a positive correlation was observed between ADCC activity and binding affinity, FCGR3A transcript levels, and surface CD16 expression levels. These results suggest that the structure and expression of the CD16 molecule differs among FcγRIIIa-V158F genotypes, and the FcγRIIIa-V158F polymorphism may be represent a haplotype with other SNPs in regulatory regions in Japanese subjects.

[Required education for medical technologists of the next generation--chairmen's introductory remarks].

This symposium is named "Required education for medical technologists of the next generation", and is held under the joint sponsorship of the Japanese Association of Medical Technology Education. We received lectures by two speakers from the clinical field and two speakers from a medical technology educational institution. From the perspective of the medical technology education institution, we were introduced to education that is actually being provided. From the clinical perspective, we were given lectures that concretely discussed the necessary role of medical technologists in clinical practice. All of the lectures were very interesting and helpful.

The evaluation of histo-blood group ABO typing by flow cytometric and PCR-amplification of specific alleles analyses and their application in clinical laboratories.

ABO antigens are oligosaccharide antigens, and are widely distributed on red blood and tissue cells as well as in saliva and body fluid. Therefore, these antigens are important not only for blood transfusion, but also for tissue cell and organ transplantations. Also, blood, hair, and seminal fluid are important sources of evidence at crime scenes, and these antigens are some of the most important markers for personal identification in forensic investigations. Here, we describe the development and use of quantitative analysis of A, B, and H antigens on red blood cells by employing flow cytometric analysis and the ABO genotyping method based on PCR-amplification of specific alleles (PASA) within DNA, especially from blood and saliva. In this study, flow cytometric analysis could be used to compare the differences between the expression of A and/or B and H antigens on red blood cells with various phenotypes, and the PASA method was able to determine the genotype of the type cisA(2)B(3) pedigree using only DNA extracted from saliva. These analysis methods are simple and useful for judging the ABO blood group system and genotyping, and are used widely throughout research and clinical laboratories and forensic fields.

Biological and clinical aspects of ABO blood group system.

The ABO blood group was discovered in 1900 by Austrian scientist, Karl Landsteiner. At present, the International Society of Blood Transfusion (ISBT) approves as 29 human blood group systems. The ABO blood group system consists of four antigens (A, B, O and AB). These antigens are known as oligosaccharide antigens, and widely expressed on the membranes of red cell and tissue cells as well as, in the saliva and body fluid. The ABO blood group antigens are one of the most important issues in transfusion medicine to evaluate the adaptability of donor blood cells with bone marrow transplantations, and lifespan of the hemocytes.This article reviews the serology, biochemistry and genetic characteristics, and clinical application of ABO antigens.

Divergence of p33/34 gene of Theileria found in Cervus nippon in Japan.

The 18S rRNA gene and the piroplasm major immunodominant protein gene (p33/34) of Theileria from various subspecies of sika deer in 8 different locations of Japan were analyzed. The similarity between 633 bp partial sequences of the 18S rRNA gene among various subspecies of sika deer was found to be between 99.7% and 100%. While the percent identities of the 412 bp partial p33/34 gene sequence and deduced amino acid sequences between Theileria of sika deer from Yamaguchi Prefecture and those found in deer from other Prefectures, were comparatively low, 68.7% to 70.1% and 64.1% to 70.0% respectively. These findings suggest that there are at least two genetically distinct strains of Theileria of sika deer in Japan.

Phylogenetic analysis of Theileria sp. from sika deer, Cervus nippon, in Japan.

The 18S rRNA genes of Theileria species detected in sika deer, Cervus nippon centralis in Yamaguchi and Cervus nippon yesoensis in Hokkaido, were analyzed. The percent identities of the nucleotide sequences of Theileria from Cervus nippon centralis and Cervus nippon yesoensis were more than 99%. The percent identities of the Theileria sp. from sika deer and Theileria sergenti, Theileria buffeli and Theileria cervi were 97, 96 and 95%, respectively. Phylogenetic analysis of the gene sequences also revealed that Theileria sp. detected from sika deer comprise a clade that is clearly distinct from the clade comprised of Theileria from cattle.

Multidrug resistance in hematological malignancy.

The recent treatment of hematological malignancies appears to be unsatisfactory in child and adult patients with acute myeloid leukemia and adult patients with acute lymphocytic leukemia. A major problem in the treatment of leukemia is caused by the development of drug resistance to chemotherapeutic agents, which is already present at diagnosis or after chemotherapy as a minimal residual disease, their resistance having originated from genetic or epigenetic mutations during prior growth of the leukemia clone. It was suggested that the mechanisms of drug resistance consist of drug resistance proteins, which work as a drug efflux pump. These are the permeability-related glycoprotein (P-Gp), the multidrug-resistance associated protein (MRP), the lung resistance protein (LRP), and other MDR proteins such as the transporter associated with antigen processing (TAP), anthracyclin resistance associated protein (ARA), MRP 2-7, and breast cancer resistance protein (BCRP). In addition, anti-apoptosis mechanisms, alterations of tumor suppressor genes, altered immunogenicity, drug resistance mechanisms for individual drugs, and clinical risk factors such as white blood cell count, age, and other factors have been reported to act in drug resistance singly or in combinations. Here we describe the update of research on the biology of MDR in the hematological malignancies and also discuss how to overcome MDR and adapt the updated treatment methods in the clinical medical field.

Expression levels of H-type alpha(1,2)-fucosyltransferase gene and histo-blood group ABO gene corresponding to hematopoietic cell differentiation.

BACKGROUND: The expression of ABO antigens on the surface of RBCs is regulated by ABO gene-encoded ABO transferase activity after the formation of the H antigen. The molecular mechanisms that control the expression of the ABO blood group antigens along with erythroid differentiation are one of the most important subjects of study in transfusion science. STUDY DESIGN AND METHODS: FUT1(H), ABO, and beta 2-microglobulin mRNA were determined by semiquantitative RT-PCR from 27 hematopoietic cultured cell lines showing various differentiation stages and cell lineages and normal PBMNCs. The expression level of each cell was analyzed with computer software (Image, NIH) and was shown as the ratio of ABO mRNA or H mRNA to beta 2-microglobulin mRNA. The H antigen was also determined by immunocytochemical methods with flow cytometry in some cell lines. RESULTS: The highest expression of H mRNA was found in KOPM-28 and HEL cell lines and a lower expression was found in the mature cells. In contrast, it was observed that H antigen expression began at the level of HEL and PL-21 cells and increased with cell maturation. The highest expression of ABO mRNA was found in K-562 and KOPM-28 cell lines and it decreased along with cell maturation. CONCLUSION: Based on these results, it is concluded that the transcription of both H and ABO genes starts early in immature peripheral blood progenitor cells but gradually decreases during cellular maturation and also that the H antigen maintains a high level of expression thereafter. These results may reflect the active synthesis of H and ABO antigens in normal hematopoietic peripheral blood progenitor cells.