Reference intervals of red blood cell parameters and platelet count for healthy adults in Japan.

Although it is known that red blood cell (RBC) parameters and platelet count depend on ethnicity and sex, their reference intervals in healthy Asian populations are limited. The aim of this study was to establish reference intervals for RBC parameters and platelet count for healthy adults in Japan. A total of 750 healthy adults (447 women and 303 men; median age 40 years (18-67 years) at seven Japanese centers who participated in regular medical checkups entered this study. Their RBC parameters and platelet count were measured using automated hematocytometers. The reference intervals of the RBC parameters and platelet count according to sex in healthy adults were determined. There was an age-specific decrease in RBC counts and an age-specific increase in mean corpuscular volume in men. This study emphasizes the need to consider sex and age in the clinical use of reference intervals of RBC parameters.

Reference intervals of white blood cell parameters for healthy adults in japan.

INTRODUCTION: While white blood cell (WBC) parameters have been suggested to depend on ethnicity and gender, reference intervals in healthy Asian populations are limited. The present study established reference intervals of WBC parameters for healthy adults in Japan. METHODS: A total of 750 healthy adults (447 women and 303 men; 18-67 years old, median 40 years old) at 7 Japanese centers who participated in regular medical checkups entered this study. The WBC parameters were measured using automated hematocytometers and blood film reviews by a manual microscopic examination. RESULTS: The reference intervals of the WBC parameters according to gender in healthy adults were determined. Age-specific decreases in WBC counts of both gender groups and in neutrophil counts of women were noted. Favorable correlations between the hematocytometer and microscopic methods were found in neutrophils, lymphocytes, and eosinophils but not in monocytes or basophils. CONCLUSION: This study suggests the need to consider gender and age in the clinical use of reference intervals of WBC parameters.

Association between cystatin C gene polymorphism and the prevalence of white matter lesion in elderly healthy subjects.

Cystatin C (CST3) is a cysteine protease inhibitor abundant in the central nervous system, and demonstrated to have roles in several pathophysiological processes including vascular remodeling and inflammation. Previously, we showed a relation of CST3 gene polymorphisms with deep and subcortical white matter hyperintensity (DSWMH) in a small case-control study. In this study, we aimed to investigate the relation in a larger cross-sectional study. Participants of a brain health examination program were recruited (n = 1795) in the study, who underwent routine blood tests and cognitive function tests. Cerebral white matter changes were analyzed by MRI. Additionally, 7 single nucleotide polymorphisms (SNPs) (-82G/C, -78T/G, -5G/A, +4A/C, +87C/T, +148G/A and +213G/A) in the promoter and coding regions of CST3 gene were examined. Among them, carriers of the minor allele haplotype -82C/+4C/+148A were significantly associated with decreased CST3 concentration in the plasma. Unadjusted analysis did not show significant relation between carriers of the minor allele haplotype and periventricular hyperintensity (PVH), but DSWMH was marginally (p < 0.054) increased in this group. After adjusting the effects of other variables like age and kidney function, logistic regression analysis revealed that carriers of the minor allele haplotype were at a significantly increased risk of developing both PVH and DSWMH. Thus, our results suggest that carriers of the minor allele haplotype -82C/+4C/+148A of CST3 gene could be at an increased risk to develop cerebral white matter disturbance.

Sub-Chronic Consumption of Dark Chocolate Enhances Cognitive Function and Releases Nerve Growth Factors: A Parallel-Group Randomized Trial.

Previous research has shown that habitual chocolate intake is related to cognitive performance and that frequent chocolate consumption is significantly associated with improved memory. However, little is known about the effects of the subchronic consumption of dark chocolate (DC) on cognitive function and neurotrophins. Eighteen healthy young subjects (both sexes; 20-31 years old) were randomly divided into two groups: a DC intake group (n = 10) and a cacao-free white chocolate (WC) intake group (n = 8). The subjects then consumed chocolate daily for 30 days. Blood samples were taken to measure plasma levels of theobromine (a methylxanthine most often present in DC), nerve growth factor (NGF), and brain-derived neurotrophic factor, and to analyze hemodynamic parameters. Cognitive function was assessed using a modified Stroop color word test and digital cancellation test. Prefrontal cerebral blood flow was measured during the tests. DC consumption increased the NGF and theobromine levels in plasma, enhancing cognitive function performance in both tests. Interestingly, the DC-mediated enhancement of cognitive function was observed three weeks after the end of chocolate intake. WC consumption did not affect NGF and theobromine levels or cognitive performance. These results suggest that DC consumption has beneficial effects on human health by enhancing cognitive function.

Physical inactivity and vitamin D deficiency in hospitalized elderlies.

Serum levels of 25(OH)D, which is known to correlate with systemic nutritional status, is used as an indicator of vitamin D sufficiency in the body. We studied the 25(OH)D status and its background factors including activity of daily living (ADL) in the elderlies hospitalized at the regional core hospital. We also examined whether or not vitamin D deficiency affects ADL among them. This study included newly hospitalized patients aged 65 years or over at Ohchi hospital April to August in 2015. At the time of admission, serum 25(OH)D concentration was measured, and ADL, instrumental ADL (IADL), cognitive function, blood test, nursing care certification were investigated as background factors. Among 209 patients, 25(OH)D was sufficient (> 30 ng/mL) only 14 cases (7%), insufficient (20-30 ng/mL) in 43 cases (20%), and deficient (< 20 ng/mL) in 152 cases (73%). Multivariate analysis showed that low ADL (OR 0.99, 95% CI 0.97-0.99) and low IADL (OR 0.88, 95% CI 0.78-0.99) were independent predictors of 25(OH)D deficiency in two models incorporating ADL and IADL, respectively. Furthermore, low 25(OH)D level was significantly associated with low ADL (OR 0.95, 95% CI 0.91-0.99) and low IADL (OR 0.93, 95% CI 0.88-0.97) scores, suggesting that vitamin D deficiency may affect physical activities. Most hospitalized elderly patients in Japan were deficient for vitamin D. In addition, physical inactivity is strongly associated with vitamin D deficiency.

Transplantation of a bone marrow mesenchymal stem cell line increases neuronal progenitor cell migration in a cerebral ischemia animal model.

Mesenchymal stem cell (MSC) transplantation is demonstrated to improve functional and pathological recovery in cerebral ischemia. To understand the underlying mechanism, we transplanted a MSC line (B10) in a rat middle cerebral artery occlusion (MCAO) model and checked the proliferation and migration of neuronal progenitor cells (NPCs). B10 transplantation increased NPCs in the subventricular zone and their migration towards the lesion area at an earlier time. Fourteen days after MCAO, some NPCs were differentiated to neurons and astrocytes. Although B10 transplantation increased total number of both astrocytes and neurons, it only increased the differentiation of NPC to astrocyte. The mRNA of polysialylation enzyme ST8SiaIV and a chemokine SDF-1 were persistently increased in B10-transplanted groups. SDF-1-positive cell number was increased in the core and penumbra area, which was expressed in macrophage/microglia and transplanted B10 cells at 3 days after MCAO. Furthermore, SDF-1 mRNA expression in cell culture was high in B10 compared to a microglia (HMO) or a neuronal (A1) cell line. B10 culture supernatant increased in vitro A1 cell migration, which was significantly inhibited by siRNA-mediated SDF-1 silencing in B10. Thus, our results suggested that MSC transplantation increased endogenous NPC migration in cerebral ischemic condition by increasing chemokine and polysialylation enzyme expression, which could be helpful for the restorative management of cerebral ischemia.

Evaluation of autonomic functions of patients with multiple system atrophy and Parkinson's disease by head-up tilt test.

The aim of this study was to evaluate the autonomic neural function in Parkinson's disease (PD) and multiple system atrophy (MSA) with head-up tilt test and spectral analysis of cardiovascular parameters. This study included 15 patients with MSA, 15 patients with PD, and 29 healthy control (HC) subjects. High frequency power of the RR interval (RR-HF), the ratio of low frequency power of RR interval to RR-HF (RR-LF/HF) and LF power of systolic BP were used to evaluate parasympathetic, cardiac sympathetic and vasomotor sympathetic functions, respectively. Both patients with PD and MSA showed orthostatic hypotension and lower parasympathetic function (RR-HF) at tilt position as compared to HC subjects. Cardiac sympathetic function (RR-LF/HF) was significantly high in patients with PD than MSA at supine position. RR-LF/HF tended to increase in MSA and HC, but decreased in PD by tilting. Consequently, the change of the ratio due to tilting (ΔRR-LF/HF) was significantly lower in patients with PD than in HC subjects. Further analysis showed that compared to mild stage of PD, RR-LF/HF at the supine position was significantly higher in advanced stage. By tilting, it was increased in mild stage and decreased in the advanced stage of PD, causing ΔRR-LF/HF to decrease significantly in the advanced stage. Thus, we demonstrated that spectral analysis of cardiovascular parameters is useful to identify sympathetic and parasympathetic disorders in MSA and PD. High cardiac sympathetic function at the supine position, and its reduction by tilting might be a characteristic feature of PD, especially in the advanced stage.

Enhanced Feedback-Related Negativity in Alzheimer's Disease.

Alzheimer's disease (AD), the most common cause of dementia in the elderly, results in the impairment of executive function, including that of performance monitoring. Feedback-related negativity (FRN) is an electrophysiological measure reflecting the activity of this monitoring system via feedback signals, and is generated from the anterior cingulate cortex. However, there have been no reports on FRN in AD. Based on prior aging studies, we hypothesized that FRN would decrease in AD patients. To assess this, FRN was measured in healthy individuals and those with AD during a simple gambling task involving positive and negative feedback stimuli. Contrary to our hypothesis, FRN amplitude increased in AD patients, compared with the healthy elderly. We speculate that this may reflect the existence of a compensatory mechanism against the decline in executive function. Also, there was a significant association between FRN amplitude and depression scores in AD, and the FRN amplitude tended to increase insomuch as the Self-rating Depression Scale (SDS) was higher. This result suggests the existence of a negative bias in the affective state in AD. Thus, the impaired functioning monitoring system in AD is a more complex phenomenon than we thought.

Single nucleotide polymorphisms of the DGKB and VCAM1 genes are associated with granulocyte colony stimulating factor-mediated peripheral blood stem cell mobilization.

We previously reported the association between LDL cholesterol level (LDL-C) and granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood (PB) hematopoietic stem cells (HSC). In this study, we investigated the association between gene single nucleotide polymorphisms (SNPs) involved in hematopoiesis and lipid level and PBHSC mobilization. In 46 patients who underwent peripheral blood stem cell harvest (PBSCH), we measured CD34-positive cells in PB and PBSCH, and the patients were classified into good, intermediate, or poor mobilizer groups based on the CD34-positive cell counts. And SNPs of the OR4C12, ENO1, RERE, DGKB, DSC3, VCAM1, CD44, and FADS1 genes were investigated. The frequency of the TT type of the DGKB gene was higher in the poor mobilizer group compared to other groups (p<0.05), whereas that of the CC type of the VCAM1 gene was high in the good mobilizer group (p<0.05). Association with the efficiency of HSC mobilization to PB were found in the SNPs of the DGKB gene involved in cell transport and SDF-1-induced migration ability and of the VCAM1 gene which is essential for HSC homing, suggesting that SNPs involved in cell migration ability might be partly involved in HSC mobilization to PB.

Low-density lipoprotein as a biomarker for the mobilization of hematopoietic stem cells in peripheral blood.

The predictor of hematopoietic stem cells (HSCs) mobilized in peripheral blood (PB) remains unknown. We retrospectively examined the relationship between serum cholesterol level and CD34-positive cells mobilized with granulocyte stimulating factor in PB. PB- mobilized CD34-positive cells were significantly higher in patients with high titers of total cholesterol and low-density lipoprotein cholesterol (LDL-C) than in patients with normal levels (average total cholesterol, 122.94 vs. 51.03/µL, p<0.05; average LDL-C 130.07 vs. 53.77, p<0.05). Multivariable analysis showed that LDL-C significantly influenced PB-mobilized CD34-positive cells, suggesting that LDL-C may be an effective biomarker for mobilization of HSCs in PB.

Therapy-related Ph+ leukemia after both bone marrow and mesenchymal stem cell transplantation for hypophosphatasia.

Bone marrow (BM) transplantation (BMT) is one of the treatment strategies for congenital metabolic disease, but leukemia secondary to intensive cytoreductive treatment is a major concern. Besides BM cells, mesenchymal stem cells (MSC) are also used for transplantation. An 8-month-old girl with hypophosphatasia underwent transplantation of haploidentical BM cells followed by two transplants of MSC obtained from her father to facilitate osteogenesis. Fludarabine(Flu)/cyclophosphamide (CPA)/anti-thymocyte globulin were used for myeloablative conditioning, but the patient developed therapy-related leukemia harboring t(9;22)(q34;q11.2); minor BCR-ABL (t-leukemia with Ph) at the age of 32 months. At the age of 40 months she underwent a second BM and third MSC transplant from the same donor. Thereafter, she achieved complete histological and molecular remission. The present case suggests that the combination of cytotoxic agents (Flu/CPA) and MSC led to t-leukemia with Ph as a consequence of chromosome instability and suppression of host anti-tumor immunity.

[Relativity in multiple myeloma and KL-6].

KL-6 is a high-molecular-weight mucinous glycoprotein discovered as a pulmonary adenocaricinoma related antigen. Its levels are used as a biomarker of lung injury in interstitial pneumonia. We here report a case of multiple myeloma with Bence-Jones lambda type whose serum KL-6 level was revealed high at a concentration of 19,400 U/ml. Next, we analyzed the blood test profiles and the concentrations of KL-6 in 20 patients with multiple myeloma. CD19/CD56 double negative fraction on myeloma cells with high expression of CD38 was found in all 5 patients with multiple myeloma having elevated KL-6 level. Patients with interstitial pneumonia show high level of KL-6. We, therefore, need to differentiate the interstitial pneumonia and the above-mentioned multiple myeloma when serum KL-6 level is high.

[Approach for the improvement of microscopic urinary sediment examination among the hospitals in using multifocal virtual software].

Microscopic examination is usually used for analysis of urinary sediment. The problem of it is difference of the examination results among hospital's clinical laboratories. Photo-survey, training conference using photographs and seminars are held for each examiner to obtain accurate results. However, diagnosis of urinary sediment with photographs is difficult because detailed 3-dimensional cell morphology of urinary sediment cannot be analyzed only with photographs. It is also difficult to have opportunity for all examiners to get enough times of real microscopic training. To evaluate the availability of multi-focal virtual software, diagnosis of urinary sediment under 17 hospital's laboratories was compared between multifocal virtual software survey and conventional photo-survey. Questionnaires were also filled out by the examiners. The rate of correct answers to virtual software survey was 73.9% that was almost same as the result of the survey by Saitama Sightron Society. The rate of correct answers by virtual software survey was higher than photo survey. As a result of questionnaires, the participants take certain advantages of virtual software in spite of several problems. In conclusion, multifocal virtual software enables an accurate diagnosis of urinary sediment in 3-dimension, which leads to reduce the discrepancies of examination results among laboratories.

Cyclopamine induces eosinophilic differentiation and upregulates CD44 expression in myeloid leukemia cells.

Cyclopamine, a plant-derived steroidal alkaloid, inhibits the hedgehog (Hh) signaling pathway by antagonizing Smoothened. This drug can induce the differentiation of myeloid leukemia cell lines and acute myeloid leukemia (AML) cells in primary culture. The treated cells were stained with Luxol-fast-blue, which is specific for eosinophilic granules. Ligation of CD44 with some specific monoclonal antibodies can reverse the differentiation of AML cells. Combined treatment with cyclopamine and a monoclonal antibody to ligate CD44 more than additively induced the differentiation of HL-60 cells. These results may provide useful information for the development of a CD44-targeted therapy in AML.

Effective ex vivo expansion of hematopoietic stem cells using osteoblast-differentiated mesenchymal stem cells is CXCL12 dependent.

Effective ex vivo expansion of hematopoietic stem cells (HSCs) is a prerequisite for HSC transplantation. Growth and maintenance of HSC is dependent on cytokine and niche factors. We investigated whether mesenchymal stem cells (MSCs) or osteogenic cytokine-differentiated MSCs play a role in HSC expansion. We used the human HM3.B10 (B10) MSC cell line and the osteoblast-differentiated B10 (Ost-B10) as a feeder layer and examined ex vivo expansion of CD34(+)CD38(-) HSCs obtained from peripheral blood (PB) and cord blood (CB) with or without several growth cytokines. Both undifferentiated B10 and Ost-B10 cells exhibited similar effects on total HSC expansion; however, Ost-B10 demonstrated a higher potency in CD34(+)CD38(-) cell-specific proliferation in the presence of cytokines compared to undifferentiated B10 HSCs. Colony-forming cell assay and long-term culture initiating cell assay revealed that Ost-B10 displayed multipotent differentiation and enabled long-term ex vivo culture of HSCs. We next examined the relationship between HSC expansion and the presence of various chemokines. CXCL4 and CXCL12 expression were increased in Ost-B10 cells compared with the B10 cells. CD34(+)CD38(-) cells were significantly increased with CXCL12, but not CXCL4 treatment. siRNA inhibition of CXCL12 decreased CXCL12 secretion in both B10 and Ost-B10, whereas expansion of CD34(+)CD38(-) cells was decreased in Ost-B10 alone. These results demonstrated that ex vivo expansion of HSCs may be highly effective through osteoblast-differentiated MSCs acting as a feeder layer, and likely operates through the CXCL12 chemokines signaling pathway.

Pure red cell precursor toxicity by linezolid in a pediatric case.

Linezolid (LZD)-induced myelosuppression has been reported in adults; however, LZD-induced pure red cell precursor toxicity rarely occurs. A 2-year-old boy diagnosed with infective endocarditis by Streptococcus mitis received LZD after developing resistance to multiple antibiotics. Although his infective symptoms were improved by LZD, progressive anemia was noticed 2 weeks after LZD therapy. Four weeks after LZD administration, his hemoglobin level was 6.5 g/dL and reticulocytes less than 0.1%. Bone marrow examination revealed markedly decreased erythropoiesis with cytoplasmic vacuolation of erythroblasts. Anemia recovered 19 days after cessation of LZD. Elevated protoporphyrin and a high LZD level in the blood suggested that mitochondrial disturbance by high-dose and long-term treatment with LZD may have been responsible for LZD-induced pure red cell precursor toxicity.

Neonatal isoimmune thrombocytopenia caused by type I CD36 deficiency having novel splicing isoforms of the CD36 gene.

Neonatal alloimmune thrombocytopenia (NAIT) occurs because of transplacentally acquired maternal platelet alloantibodies. Most of the alloantibodies are against human platelet antigens, but the alloantibody against CD36 is rare. A full-term female baby was delivered by a mother who experienced two spontaneous abortions. The baby had thrombocytopenia with cephalhematoma. The platelet count increased by immunoglobulin therapy (400 mg/kg) for 3 d. Platelet antibody was detected in the postpartum maternal serum. The specificity of the antibody directed against platelets was identified as anti-Nak(a) (CD36). Flow cytometric analysis showed no expression of CD36 in both platelets and monocytes from mother. Mutation analysis revealed two different splicing isoforms of maternal CD36 mRNA. One allele was exon 4 skipping, another was exon 9 skipping, both of which led to a frameshift and produced a truncated CD36 protein. These results indicate that NAIT is caused by maternal CD36 deficiency having CD36 splicing abnormalities.

Immortalized human microglial cell line: phenotypic expression.

Microglia are a major neuroglial component of the CNS, playing an important role as resident immunocompetent and phagocytic cells in the CNS in the event of injury and disease. To understand the role of microglia in the CNS in health and diseases, we have recently established an immortalized clonal cell line of human microglia, HMO6, from human embryonic telencephalon tissue by using a retroviral vector encoding v-myc. This immortalized microglia HMO6 cell line exhibits cell-type-specific antigens for microglia, including CD11b (Mac-1), CD68, CD86 (B7-2), HLA-ABC, HLA-DR, and RCA-1 lectin, and actively phagocytoses latex beads.