Differences in serum and plasma levels of microRNAs and their time-course changes after blood collection.

Background: Serum and plasma are used for measurements of microRNAs (miRNAs) as biomarkers of various diseases. However, no consistent findings have been obtained regarding differences in serum and plasma levels of miRNAs. The purpose of this study was to clarify differences in serum and plasma levels of total miRNAs and their time-course changes after blood collection. Methods: Venous blood was collected from healthy men, and samples were prepared at the time points of 0, 15, 30, 60 and 180 min after blood collection for plasma and after clot formation for serum. Levels of total miRNAs were analyzed by the hybridization method using the 3D-Gene miRNA Oligo chip. Results: About one third of 2632 miRNAs tested showed levels high enough for comparison of serum and plasma levels and for investigation of their time-course changes. Levels of 299 miRNAs at time 0 were significantly different in serum and plasma. Levels of representative platelet-derived miRNAs including miR-185-5p, -22-3p and -320b were significantly higher in plasma than in serum, while levels of representative erythrocyte-derived miRNAs including miR-451a, -486-5p and -92a-3p were not significantly different in serum and plasma. Plasma levels of 173 miRNAs and 6 miRNAs showed significant decreasing and increasing tendencies, respectively, while there were no miRNAs in serum that showed significant time-course changes. Conclusion: The results suggest that careful attention should be paid when comparing serum and plasma levels of miRNAs and that plasma samples should be prepared early after blood collection.

Augmentation by resveratrol of the inhibitory effect of ethanol on platelet aggregation.

Ethanol and resveratrol have been shown to inhibit platelet aggregation. The aim of this study was to determine whether resveratrol has an additional effect on ethanol-induced inhibition of platelet aggregation. Ca2+ entry and subsequent aggregation of human platelets were measured by the fluorescence method and light transmittance method, respectively. Thromboxane B2 concentrations in media containing platelets were measured by using the enzyme-linked immunosorbent assay. Platelet aggregation induced by thrombin (0.025 U/ml) was significantly inhibited by preincubation of platelets with ethanol (0.5%). Preincubation with resveratrol (3.125 µM), which did not affect thrombin-induced platelet aggregation, significantly augmented the inhibitory effect of ethanol on platelet aggregation. Similar synergic effects of ethanol and resveratrol were found on aggregatory responses to collagen (2 µg/ml) and arachidonic acid (0.25 mM). On the other hand, the thrombin-induced increase in intracellular Ca2+ concentration ([Ca2+]i) was not affected by ethanol alone, resveratrol alone or both ethanol and resveratrol together. In nominally Ca2+-free medium, arachidonic acid (0.75 mM) caused a potent platelet aggregation, which was not affected by the presence of ethanol alone, resveratrol alone, or both of them together. Thromboxane B2 formation induced by thrombin was significantly inhibited by ethanol (0.5%) alone and resveratrol (3.125 µM) alone, and these inhibitory effects were significantly augmented in the presence of both ethanol and resveratrol together. Resveratrol shows an additive effect on ethanol-induced inhibition of platelet aggregation. This effect by resveratrol is partly explained by its inhibitory action on thromboxane A2 production in platelets. In addition, both ethanol and resveratrol attenuate platelet aggregation through acting on the Ca2+-dependent intra-platelet pathway after an increase in [Ca2+]i induced by thrombin.

Evidence for Indoxyl Sulfate as an Inducer of Oxidative Stress in Patients With Diabetes.

BACKGROUND/AIM: Indoxyl sulfate is a metabolite of tryptophan and its urinary level reflects the status of bacterial flora in the intestine. Indoxyl sulfate possesses prooxidant properties and is implicated in various diseases including chronic kidney disease and cardiovascular diseases. However, the relation of urinary indoxyl sulfate to oxidative stress is not known. PATIENTS AND METHODS: The association of urinary indoxyl sulfate levels with urinary levels of oxidative stress markers, 15-isoprostane F2t and pteridine derivatives, was investigated in 255 patients with type 2 diabetes. Indoxyl sulfate and pteridine derivatives were measured by using spectrofluorometry. RESULTS: Urinary levels of indoxyl sulfate, pteridines, and 15-isoprostane F2t showed a normal distribution after logarithmic transformation but not before it, and they were thus used for parametric analysis after logarithmic transformation. Urinary indoxyl sulfate levels were significantly correlated (p<0.01) with urinary 15-isoprostane F2t and pteridine levels [Pearson's correlation coefficients: 0.503 (15-isoprostane F2t) and 0.562 (pteridines)]. These associations were also found in multivariable analysis after adjusting for age, sex, insulin therapy for diabetes, body mass index, mean arterial pressure, hemoglobin A1c, estimated glomerular filtration rate, urinary albumin, and histories of smoking and alcohol drinking. CONCLUSION: Urinary indoxyl sulfate levels showed associations with urinary levels of oxidative stress markers, and the associations were independent of age, sex, insulin therapy for diabetes, body mass index, blood pressure, glycemic status, renal function, smoking, and alcohol drinking. Indoxyl sulfate appears to be an important determinant of redox balance in patients with diabetes.

Data on urinary pteridines as a discriminator of atherosclerotic risk in patients with diabetes.

Presented here are the supplemental data of the research article "Urinary pteridines as a discriminator of atherosclerotic risk in patients with diabetes" [1]. These data provide the first information on variables that affect urinary levels of pteridines (oxidized-form pteridine derivatives) in patients with diabetes mellitus. In linear regression analysis, gender (women vs. men), current history of smoking and urinary albumin showed significant positive correlations with pteridines, while there were significant inverse correlations of pteridines with a history of alcohol drinking and body mass index. The above associations were confirmed by using analysis of covariance and logistic regression analysis. Among the eight variables (age, gender, medication therapy for diabetes, smoking, alcohol drinking, body mass index, hemoglobin A1c and urinary albumin) tested, smoking showed the strongest association with urinary pteridines.

Relationship of urinary liver-type fatty acid-binding protein with cardiovascular risk factors in the Japanese population without chronic kidney disease: Sasayama study.

BACKGROUND: Urinary liver-type fatty acid-binding protein (L-FABP) is a well-known marker of proximal tubular impairment. We evaluated the relationship between cardiovascular disease (CVD) risk factors and levels of L-FABP in a cross-sectional community-based study. Participants with normoalbuminuria and normal estimated glomerular filtration rate (eGFR), that is, non-chronic kidney disease (non-CKD), were enrolled in this study. To the best of our knowledge, this is the first study to focus on the association between CVD risk factors and a proximal tubular marker in the Japanese general population with normoalbuminuria and normal eGFR. METHODS: The present study is part of the Sasayama study. The participants included 1000 community residents (447 men and 553 women) aged 40-64 years without a history of CVD or renal dysfunction. Out of these participants 375 men and 477 women, defined as non-CKD, were included for further analysis. In each sex, the highest quintile group was considered to have high-normal L-FABP levels. A multiple logistic regression model was used to evaluate the relationship between risk factors for CVD and high-normal L-FABP levels in the non-CKD participants. We performed a similar analysis using the high-normal urinary albumin to creatinine ratio (UACR) as a dependent variable instead of L-FABP. RESULTS: Among the non-CKD participants, in the highest quintile group (Q5, top 20%), L-FABP was ≥2.17 µg/gCre in men and ≥ 2.83 µg/gCre in women. In women, the multivariate odds ratio was 3.62 (1.45-9.00) for high-normal L-FABP in the presence of diabetes mellitus (DM) compared with that in the group without DM. However, the relationship between DM and the UACR level was not significant. In men, DM was significantly associated with high-normal UACR. However, the relationship with L-FABP levels was not significant. CONCLUSIONS: The presence of DM was more strongly related to high-normal L-FABP levels than to high-normal UACR in women even at the stage of normoalbuminuria and normal eGFR. Our results were also consistent with the findings of a previous study where women were more prone to nonalbuminuric renal impairment compared to men, although further studies are required to confirm the results.

Urinary pteridines as a discriminator of atherosclerotic risk in patients with diabetes.

Background and aims: We have recently proposed urinary pteridine level as a useful biomarker of oxidative stress in a general population. However, the significance of urinary pteridines in patients with diabetes is unknown. Methods: The relationships of the level of urinary pteridine derivatives with d-dimer, ankle-brachial pressure index (ABI), and known cardiovascular risk factors were investigated in patients with type 2 diabetes. Results: Urinary pteridine level showed significant positive correlations with urinary15-isoprostane F2t, female gender, history of smoking and d-dimer and significant inverse correlations with history of alcohol drinking, body mass index (BMI) and ABI. ABI was significantly lower and d-dimer was significantly higher in the highest tertile group of pteridines than in the lowest tertile group. The odds ratios of the highest vs. lowest tertiles for low ABI and high d-dimer were significantly higher than the reference level. The above relationships of urinary pteridines with ABI and d-dimer were not altered when age, gender, BMI, hemoglobin A1c and history of alcohol drinking were used as explanatory variables in multivariable analyses. History of smoking confounded the relation of pteridines with ABI but not that with d-dimer. However, in logistic regression analysis, the association between pteridines and ABI remained significant with adjustment for history of smoking. Conclusion: Urinary pteridine level was associated with d-dimer and ABI, which reflect blood coagulability and arterial flow to the lower extremities, respectively, and is thus thought to be a useful discriminator of thromboatherosclerotic risk in patients with diabetes.

Resveratrol inhibits Ca2+ signals and aggregation of platelets.

BACKGROUND: Resveratrol has been shown to inhibit platelet aggregation. However, the mechanism for this action of resveratrol remains to be clarified. The purpose of this study was to elucidate the Ca2+-related mechanism for the inhibitory action of resveratrol on platelet aggregation. METHODS: Ca2+ entry and subsequent aggregation of human platelets induced by different stimulants including thrombin, thapsigargin, and 1-oleoyl-2-acetylglycerol (OAG) were measured by the fluorescence method and light transmittance method, respectively. Each stimulant was added to a nominally Ca2+-free medium containing platelets, and then CaCl2 was added to the medium to induce Ca2+ influx into platelets. RESULTS: Thapsigargin-induced Ca2+ entry into platelets and subsequent platelet aggregation were significantly inhibited in the presence of resveratrol at 6.25 µM or higher concentrations, while OAG-induced Ca2+ entry and subsequent platelet aggregation were not affected by resveratrol at concentrations up to 50 µM. In the nominally Ca2+-free medium, thrombin induced a small transient increase in intracellular Ca2+ concentrations, which was attenuated in the presence of resveratrol at 12.5 µM or higher concentrations. Thrombin-induced Ca2+ entry into platelets and subsequent platelet aggregation were significantly inhibited in the presence of resveratrol at 12.5 µM or higher concentrations. CONCLUSIONS: The results suggest that resveratrol inhibits thrombin-induced platelet aggregation through decreasing Ca2+ release from its stores and inhibiting store-operated Ca2+ influx into platelets.

Inverse association between habitual alcohol drinking and d-dimer in patients with type 2 diabetes mellitus.

Alcohol is known to inhibit blood coagulation. Patients with diabetes mellitus are prone to show hypercoagulability. However, it remains to be clarified whether and how habitual alcohol drinking affects coagulability in patients with diabetes. The purpose of this study was to determine the relationship between alcohol intake and d-dimer, a sensitive marker of blood coagulation, in patients with diabetes. We investigated the relationship between alcohol intake and d-dimer in plasma of 269 patients with type 2 diabetes by using analysis of covariance and logistic regression analysis after adjustment for age, gender, body mass index, hemoglobin A1c, and histories of smoking and anti-coagulation therapy. Log-transformed d-dimer and HDL cholesterol were significantly lower and higher, respectively, in regular drinkers than in nondrinkers, while there were no significant differences in log-transformed d-dimer and HDL cholesterol in occasional drinkers and nondrinkers. Odds ratios of regular drinkers vs. nondrinkers for high d-dimer (0.46 [0.21-0.98]) and low HDL cholesterol (0.20 [0.08-0.50]) were significantly lower than the reference level, while the odds ratios of occasional drinkers for high d-dimer (1.24 [0.41-3.73]) and low HDL cholesterol (0.43 [0.15-1.25]) were not significantly different from the reference level. HDL cholesterol showed a significant inverse correlation with log-transformed d-dimer both in overall subjects and in nondrinkers. Regular drinking, but not occasional drinking, was associated with lower d-dimer levels, suggesting that habitual alcohol drinking suppresses hypercoagulability in patients with diabetes. There is an alcohol intake-independent inverse association between HDL cholesterol and d-dimer.

Inhibition by Ethanol of Shear Stress-Induced Formation of Platelet Thrombi in Whole Blood.

AIMS: The risk of thromboatherosclerotic disease is lower in moderate drinkers than in non-drinkers. We investigated the effects of ethanol on platelet aggregation under a condition with shear stress. SHORT SUMMARY: Shear stress-induced formation of platelet thrombi is inhibited by ethanol at its attainable concentrations after drinking. This effect is prominent at the early stage of thrombus formation, being in agreement with inhibitory actions of ethanol on the initial steps of platelet activation such as Ca2+ entry and phospholipase A2 activation. METHODS: Platelet aggregation was evaluated by using a total thrombus-formation analysis system, and shear rates of 1000 s-1 (low), 1500 s-1 (middle) and 2000 s-1 (high) were loaded to whole blood. The times required to generate increases in flow pressure of 10 kPa (T10), 30 kPa (T30) and 50 kPa (T50) in microchips containing the blood, which depend on the degree of thrombus generation, were recorded. RESULTS: Under the conditions of the low-grade and middle-grade shear rates, T10 and T30 were significantly longer in the presence of ethanol at 0.25-1% than in the absence of ethanol. T10 under the condition of the low-grade shear rate and T30 under the conditions of the low-grade and middle-grade shear rates were also significantly longer in the presence of ethanol at 0.125% than in the absence of ethanol. On the other hand, T50 under the conditions of the low-grade and middle-grade shear rates was not significantly different in the absence and presence of ethanol at 0.125, 0.25 and 0.5%. Under the condition of the high-grade shear rate, T10, T30 and T50 were not significantly different in the absence and presence of ethanol at its lower concentrations. CONCLUSIONS: Ethanol at attainable concentrations inhibits platelet thrombus formation induced by shear stress, and the inhibitory effect of ethanol is prominent at the early stage of thrombus formation.

Relationships of oxidized HDL with blood coagulation and fibrinolysis in patients with type 2 diabetes mellitus.

Although oxidization of LDL is known to be a crucial step for atherosclerotic progression, the significance of oxidized HDL remains to be clarified. The purpose of this study was to determine the relationships of oxidized HDL with blood coagulation and fibrinolysis in patients with diabetes. The subjects were outpatients with type 2 diabetes (n = 163; median hemoglobin A1c, 6.9%). Activities of blood coagulation and fibrinolysis were evaluated by levels of thrombin-anti-thrombin complex (TAT) and plasmin-α2 plasmin inhibitor complex (PIC), respectively. Relationships of oxidized HDL with TAT and PIC were investigated by using linear regression analysis and logistic regression analysis. Oxidized HDL showed a significant inverse correlation with TAT and a marginally significant correlation with PIC (Spearman's rank correlation coefficient: TAT, - 0.205 [p < 0.01]; PIC, - 0.135 [p = 0.087]). Prevalence of high TAT was significantly lower in the 3rd tertile group for oxidized HDL than in its 1st tertile (20.4 vs. 5.6%, p < 0.05), and prevalence of high PIC was marginally significantly lower in the 3rd tertile group for oxidized HDL than in its 1st tertile (40.7 vs. 24.1%, p = 0.099). In multivariate logistic regression analysis using age, gender, smoking, alcohol drinking, BMI, hemoglobin A1c, therapy for dyslipidemia, therapy for diabetes and anti-coagulation therapy as explanatory variables, odds ratios for high TAT and high PIC in the 3rd tertile group for oxidized HDL versus its 1st tertile group were significantly lower than the reference level of 1.00 (high TAT: 0.19 [0.04-0.99], p < 0.05; high PIC: 0.33 [0.12-0.95], p < 0.05). The frequency of high TAT or high PIC was lower in the higher tertile group for oxidized HDL than in its lower tertile group. Thus, oxidized HDL is thought to be inversely associated with both blood coagulation and fibrinolysis in patients with type 2 diabetes.

Effects of methanol and formic acid on human platelet aggregation.

BACKGROUND: Although ethanol is known to inhibit platelet aggregation, the effects of another variant of alcohol, methanol, have not been reported. The purpose of this study was to determine whether methanol and its metabolite, formic acid, affect Ca2+ entry into and subsequent aggregation of platelets in vitro. METHODS: Ca2+ entry into and aggregation of human platelets were measured by spectrofluorometry using Fura-2/AM as an indicator and the light transmission method, respectively. RESULTS: Thrombin-induced platelet aggregation was significantly augmented by methanol at pharmacological concentrations (0.5-2%) in a concentration-dependent manner. Methanol at 2% significantly attenuated thapsigargin-induced platelet aggregation, which was not significantly affected by lower concentrations (0.5 and 1%) of methanol. Methanol (0.5-2%) did not significantly affect platelet aggregation induced by 1-oleoyl-2-acetyl-sn-glycerol (OAG), or Ca2+ entry into platelets induced by thrombin, thapsigargin, or OAG. Platelet aggregation induced by thrombin, thapsigargin, or OAG was significantly inhibited by formic acid at toxic concentrations (0.01% or higher). Ca2+ entry into platelets induced by thrombin or thapsigargin was also significantly inhibited by formic acid at 0.01% or higher, while that induced by OAG was not affected by formic acid at 0.005 and 0.01% and augmented by that at 0.02%. CONCLUSIONS: Methanol at pharmacological doses has diverse effects on platelet aggregation, depending on the aggregation stimuli, without affecting Ca2+ entry into platelets. Formic acid at toxic concentrations has an inhibitory action on platelets aggregation, which was partly explained by the reduction of Ca2+ entry into platelets.

Detection of Subclinical Peripheral Artery Ischemia in Healthy Male Smokers by an Ankle-Brachial Index After Exercise: Sasayama Study.

We investigated the relationship between smoking and the risk of nonnormal (≤0.99) ankle-brachial index (ABI) at rest and after ankle plantar flexion exercise in healthy male community dwellers. A cross-sectional study was performed including 228 Japanese men aged 40 to 64 years without a history of cardiovascular diseases. Participants were classified as never, ex-, and current smokers. We estimated the multivariate-adjusted odds ratios (ORs) for nonnormal ABI of ex- and current smokers in relation to never smokers after adjusting for age and other confounding factors. At rest, the prevalence of nonnormal ABI was not significantly different by smoking status. After exercise, the prevalence of nonnormal ABI increased from 1.8% to 11.5% in ex-smokers and from 3.8% to 17.0% in current smokers, while the prevalence did not significantly change in never smokers. The multivariate-adjusted OR for nonnormal ABI after ankle plantar flexion exercise, in relation to never smokers, was 3.85 (95% confidence interval [CI]: 0.79-18.9) for ex-smokers and 6.97 (95% CI: 1.32-36.7) for current smokers. Our results suggest that ABI after ankle plantar flexion exercise is useful for early detection of subclinical peripheral artery ischemia in male smokers without typical symptoms.

Cysteinylated transthyretin as a discriminator of cardiovascular risk in patients with diabetes mellitus.

BACKGROUND: The cysteine residue on transthyretin (TTR) is susceptible to be oxidized, and serum cysteinylated TTR (Cys-TTR) level is thought to reflect oxidative stress. The purpose of this study was to elucidate the relationship between Cys-TTR and arterial stiffness, a known predictor of cardiovascular disease, in patients with type 2 diabetes. METHODS: The subjects were 105 male outpatients with type 2 diabetes. Arterial stiffness was evaluated by measuring cardio-ankle vascular index (CAVI). The relationship between CAVI and ratio of Cys-TTR to total TTR (Cys-TTR ratio) was analyzed. RESULTS: Cys-TTR ratio was significantly correlated with CAVI (Pearson's correlation coefficient: 0.316, p<0.01), and CAVI was significantly higher in the 3rd tertile group for Cys-TTR ratio than in its 1st tertile group. These relationships were also significant after adjusting for age, smoking, alcohol drinking, body mass index, mean arterial pressure, hemoglobin A1c, LDL cholesterol-to-HDL cholesterol ratio and eGFR. Prevalence of high CAVI (≥10.0) was significantly higher in the 3rd tertile for Cys-TTR ratio than in its 1st tertile and tended to be higher with an increase in tertile (28.6% in the 1st tertile, 42.9% in the 2nd tertile and 60.0% in the 3rd tertile). Odds ratio (OR) for high CAVI of the 3rd vs. 1st tertile groups for Cys-TTR ratio was significantly higher than the reference level of 1.00 both before and after adjustment for the above cardiovascular risk factors (crude OR, 3.75 [1.38-10.17]; adjusted OR, 5.09 [1.39-18.64]). CONCLUSIONS: Cys-TTR ratio is associated with arterial stiffness in patients with diabetes and is proposed as a new discriminator of cardiovascular risk.

Selective induction of anti-inflammatory monocyte-platelet aggregates in a model of pulsatile blood flow at low shear rates.

In patients with cardiovascular abnormalities or immunological disorders, an increased number of circulating leukocyte-platelet aggregates is observed. Leukocyte-platelet aggregates play an essential role in linking the hemostatic and immune systems. High shear stress and pro-coagulant and pro-inflammatory stimulants are known to activate platelets and promote the formation of aggregates. Pulsatile blood flow under low shear stress can also induce platelet activation in comparatively mild conditions. However, the effect of such events on leukocyte-platelet aggregates has not yet been investigated. To determine whether low shear stress affects the formation of aggregates, we established a simple "inverting rotation" method of inducing periodic changes in the direction of blood flow in combination with low shear stress. We demonstrated that after the inverting rotation treatment for 10-20 min more than 70% of monocytes selectively aggregated with platelets. The formation of monocyte-platelet complexes was inhibited by an anti-CD162 (PSGL-1) monoclonal antibody or a Ca(2+) chelator. The phagocytic activity of monocytes was augmented by inverting rotation, whereas phagocytosis mediated by granulocytes remained unaffected. Interestingly, the formation of monocyte-platelet complexes suppressed the production of pro-inflammatory cytokines such as interleukin (IL)-1ß. At the same time, monocyte-platelet complexes augmented the expression of the anti-inflammatory cytokine IL-10. Our results suggest that platelet-bound monocytes show an anti-inflammatory phenotype under low shear stress conditions. Thus, our method provided new insights into the mechanisms of monocyte-platelet aggregate formation and regulation.

Potential Biomarker Peptides Associated with Acute Alcohol-Induced Reduction of Blood Pressure.

The purpose of this study was to explore the peptides that are related to acute reduction of blood pressure after alcohol drinking. Venous blood was collected from male healthy volunteers before and after drinking white wine (3 ml/kg weight) containing 13% of ethanol. Peptidome analysis for serum samples was performed using a new target plate, BLOTCHIP®. Alcohol caused significant decreases in systolic and diastolic blood pressure levels at 45 min. The peptidome analysis showed that the levels of three peptides of m/z 1467, 2380 and 2662 changed significantly after drinking. The m/z 1467 and 2662 peptides were identified to be fragments of fibrinogen alpha chain, and the m/z 2380 peptide was identified to be a fragment of complement C4. The intensities of the m/z 2380 and m/z 1467 peptides before drinking were associated with % decreases in systolic and diastolic blood pressure levels at 45 min after drinking compared with the levels before drinking, while there were no significant correlations between the intensity of the m/z 2662 peptide and % decreases in systolic and diastolic blood pressure levels after drinking. The m/z 1467 and 2380 peptides are suggested to be markers for acute reduction of blood pressure after drinking alcohol.

Association between serum long-chain n-3 and n-6 polyunsaturated fatty acid profiles and glomerular filtration rate assessed by serum creatinine and cystatin C levels in Japanese community-dwellers.

BACKGROUND: Plasma concentration of n-3 polyunsaturated fatty acids (PUFAs) has been reported to be associated with renal function in Western populations. However, few studies have investigated the association between serum long-chain n-3 and n-6 PUFA profiles and renal function in a Japanese population with high marine-derived long-chain n-3 PUFA intake. METHODS: A cross-sectional study was performed in 549 Japanese rural community-dwellers aged 40 to 64 years. In adjusted analysis of covariance, we assessed the relationship between estimated glomerular filtration rate (eGFR) and tertiles of serum long-chain n-3 and n-6 PUFA profiles ([eicosapentaenoic acid {EPA} + docosahexaenoic acid {DHA}]:arachidonic acid [AA]). GFR was estimated by Japanese specific equations using serum creatinine and cystatin C (eGFRcre and eGFRcys). Using multivariate-adjusted linear regression models, we also assessed the relationships between eGFRs and several n-3 and n-6 PUFAs, which have been suggested to be associated with renal function. RESULTS: In all participants, higher dietary fish intake as assessed by a semi-quantitative questionnaire was associated with higher serum value of (EPA+DHA):AA. Participants in the higher (EPA+DHA):AA tertiles had non-significantly higher eGFRcre and significantly higher eGFRcys (P = 0.016). In addition, eGFRcys in T2+T3 of (EPA+DHA):AA was significantly higher than that in T1 (adjusted mean eGFRcys, T1: 87 ml/min/1.73 m(2), T2+T3: 91 ml/min/1.73 m(2); P < 0.01). Among the PUFAs, only (EPA+DHA) was significantly associated with eGFRcys. CONCLUSIONS: Serum (EPA+DHA):AA, which reflects an individual's fish intake, might be associated with eGFRcys in Japanese community-dwellers.

Hairy cell leukemia accompanied by Evans syndrome.

We report a case of Western type hairy cell leukemia (HCL), a very rare leukemia in Japan. In this malignancy, leukemic cells in a peripheral blood film may be missed due in part to accompanying pancytopenia and in part to loss of typical cytoplasmic projections if prepared in a conventional Japanese way using forced air-drying. Our present patient also had a variety of autoantibodies and the clinical picture was primarily that of Evans syndrome (ES), suggesting disturbed immune responses associated with the HCL. Although HCL accompanied by either AIHA or ITP has been reported, the occurrence of ES in HCL is extremely rare.

Inhibition of thrombin-induced Ca²âº influx in platelets by R59949, an inhibitor of diacylglycerol kinase.

OBJECTIVES: The aim of this study was to determine whether diacylglycerol kinase (DGK) is involved in transplasmalemmal Ca²âº influx of platelets. METHODS: Effects of R59949, an inhibitor of diacylglycerol kinase, on intracellular Ca²âº concentration ([Ca²âº](i) ) and mRNA expression of DGK isozymes were investigated using washed human platelet suspensions. KEY FINDINGS: Thrombin-induced increase in [Ca²âº](i) was significantly inhibited by pretreatment of platelets with R59949, while thapsigargin-induced increase in [Ca²âº](i) was comparable in platelets with and without R59949 pretreatment. Thapsigargin-induced increase in [Ca²âº](i) was markedly attenuated in the presence of SKF-96365. In the presence of SKF-96365, thrombin-induced increase in [Ca²âº](i) was significantly attenuated, and additional treatment with R59949 caused a further decrease in [Ca²âº](i) . Pretreatment of platelets with 1-butanol significantly attenuated thrombin-induced increase in [Ca²âº](i) , while thrombin-induced increase in [Ca²âº](i) was augmented in the presence of propranolol. mRNA expression of DGK-α and DGK-γ, which are known to be inhibited by R59949, in platelets was confirmed by RT-PCR analysis. CONCLUSIONS: R59949 inhibited a store-depletion-insensitive component of transplasmalemmal Ca²âº entry induced by thrombin, while store-operated Ca²âº entry was not affected by R59949. The results of this study suggest that phosphatidic acid is involved in thrombin-induced Ca²âº influx of platelets.