Association of platelet response to cilostazol with clinical outcome and CYP genotype in patients with cerebral infarction.

INTRODUCTION: Cilostazol, an anti-platelet drug that inhibits phosphodiesterase 3, is beneficial for patients with atherothrombosis. In contrast to other anti-platelet drugs such as aspirin and thienopyridines, little information is available on the relationship between platelet responses to cilostazol and clinical outcomes. MATERIALS AND METHODS: We conducted a prospective study on patients with cerebral infarction who were treated with cilostazol. The platelet response to cilostazol was assessed with our new assay for the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) subsequent to the pharmacological action of cilostazol. Patients were followed up for 2 years and the relationship between VASP assay results and the recurrence of thrombotic events was examined. We also investigated the effects of CYP3A5 and CYP2C19 genotypes involved in the metabolism of cilostazol on the platelet response to cilostazol. RESULTS: Among the 142 patients enrolled, 130 completed the 2-year follow-up and the recurrence of thrombotic events was noted in 8 (6.2%). VASP phosphorylation levels were significantly lower in patients with than in those without recurrence. The combined genotype of CYP3A5*1/*3 and CYP2C19*1/*1 was associated with a low level of VASP phosphorylation, while either genotype was not. A multivariate analysis showed that high residual platelet reactivity during the cilostazol treatment, which was defined by a low response of platelet VASP phosphorylation to cilostazol, was an independent risk factor for the recurrence of thrombotic events. CONCLUSION: A low platelet response to cilostazol determined by a new platelet assay was associated with the recurrence of thrombotic events in patients with cerebral infarction.

Asymptomatic pericardial effusion in patients with systemic lupus erythematosus.

To determine the frequency and clinical correlates of asymptomatic pericardial effusion (PE) in patients with systemic lupus erythematosus (SLE), echocardiography and electrocardiography were performed in 50 consecutive patients with SLE. Among 50 patients with SLE, 12 patients (24%) had PE and 17 patients (34%) had hypoalbuminaemia. Patients with PE had a significantly lower serum albumin (P < 0.001), higher incidence of proteinuria (P = 0.003), higher C-reactive protein (P = 0.036) and pulmonary artery systolic pressure (P = 0.011) and tended to have a higher incidence of PR-segment depression (P = 0.082) compared with those without PE. When four variables (PR-segment depression, C-reactive protein, serum albumin and pulmonary artery systolic pressure) were used in the multivariate analysis, serum albumin (P = 0.005, odds ratio = 0.016) and pulmonary artery systolic pressure (P = 0.010, odds ratio = 1.106) emerged as significant variables related to the occurrence of asymptomatic PE. Thus, an increase in hydrostatic pressure of the right heart cavities and a decrease in colloid osmotic pressure were important factors associated with the presence of asymptomatic PE in patients with SLE.

Stenting procedure for sinus stenosis with transverse-sigmoid dural arteriovenous fistulas. A case report.

SUMMARY: We reported the dural AVF case with sinus stenosis, that was entirely treated through the stenting procedure. 61-year-old male had been realizing the attack which causes bilateral visual problem. He would have suffered from the intracranial hypertension caused by dural AVF in the right transverse sinus and left transverse sinus stenosis.We performed TVE and sinus stenting, then used the antiplatelet and the anticoagulant. However, six months later, he suffered from SAH due to recurrence of dural AVF. We performed TVE again, denser packing than usual. Two years later, he have no symptom, angiographically, there was no recurrence of dural AVF and patency of stented sinus. We think denser embolizations should have performed in case of dural AVF with sinus stenting.

Polymorphisms in the 5'-upstream region of the PKCbeta gene in Japanese patients with Type 2 diabetes.

AIMS: Protein kinase C (PKC), a serine/threonine kinase, is known to be activated in various tissues under hyperglycaemic conditions. Notably, PKCbeta, a member of the conventional PKC group, is the predominant isoform detected in vascular tissues and could be involved in the development of diabetic vascular complications. In the present study, we investigated genetic variations in the 5'-upstream region of the PKCbeta gene to assess their possible relation to vascular complications in diabetic patients. METHODS: Variations upstream from the PKCbeta gene (-1066/+256) were examined in 60 Type 2 diabetic patients using a cycle sequencing method. Screening of detected variations was performed in 204 Type 2 diabetic patients and 160 healthy controls. RESULTS: Five single nucleotide polymorphisms; C(-238)G, C(-287)T, A(-348)G, C(-546)G, and C(-853)T, were identified in the upstream region. The C(-287)T and A(-348)G polymorphisms were in perfect linkage disequilibrium. There were no significant differences in genotype or allele frequencies of the five polymorphisms among the diabetic patients and healthy subjects. However, both -238GG and -287CC (-348GG) homozygotes showed significantly higher frequencies of macrovascular disease compared with patients with other genotypes. Further, an electrophoretic mobility shift assay revealed that the -238G fragment had a five-fold higher affinity for transcription factor Sp1 when compared with -238C. CONCLUSIONS: The C(-238)G and C(-287)T-A(-348)G polymorphisms in the 5'-upstream region of the PKCbeta gene may have an effect on the susceptibility of diabetic vascular complications through an alteration of tissue PKCbeta density or function.

A new labyrinthulid isolate, which solely produces n-6 docosapentaenoic acid.

A labyrinthulid strain, L59, was isolated from a leaf floating on seawater collected at the coastal area of Hokkaido Prefecture, Japan. Strain L59 contained only n-6 docosapentaenoic acid ( n-6 DPA) among all the long-chain polyunsaturated fatty acids. The proportion of n-6 DPA in the total fatty acids was 48.1% and the total fatty acids content in the cell dry weight was 26.6%. Many oil bodies were observed in the cell, mostly in the vicinity of cell membranes. The strain had spindle-shaped cell bodies and all cells were surrounded by ectoplasmic net elements. It was also clearly classified in the labyrinthulid group by phylogenetic analysis. In the optimum culture condition, using soybean oil and peptone as carbon and nitrogen sources, 0.53 g of n-6 DPA/l was produced at 20 degrees C in 7 days.

Transcriptional regulation of serum amyloid A1 gene expression in human aortic smooth muscle cells involves CCAAT/enhancer binding proteins (C/EBP) and is distinct from HepG2 cells.

Regulation of acute-phase serum amyloid A (A-SAA) synthesis by proinflammatory cytokines and steroid hormones in human aortic smooth muscle cells (HASMCs) is distinct from that in HepG2 cells. To study the cis- and trans-activating promoter element involved in the SAA1 gene expression by HASMCs and HepG2 cells, we constructed plasmid vectors for luciferase reporter gene assay with varying lengths of SAA1 upstream regulatory region (up to 1431 bp), and examined their response to proinflammatory cytokines and/or steroid hormones. The corresponding vectors with the SAA4 upstream regulatory region served as controls. The presence of proposed transcriptional regulatory factors binding to these regions was confirmed immunohistochemically. The sequences of 1478 and 1836 bp of the SAA1 and SAA4 5'-flanking regions were determined, respectively. SAA1 promoter transcription in cultured HASMCs was upregulated not by proinflammatory cytokines, but rather by glucocorticoids. This differed from HepG2 cells, in which SAA1 promoter transcription was upregulated synergistically by proinflammatory cytokines and glucocorticoids. The promoter activity of a series of truncated SAA1 promoter constructs measured using the reporter gene assay showed that the 5'-region from -252 to -175, containing a consensus site for CCAAT/enhancer binding proteins alpha,beta (C/EBPalpha,beta), was essential for SAA1 induction in HASMCs. In HepG2 cells, the 5'-region from -119 to -79, containing a nuclear factor kappa-B (NFkappaB) consensus sequence, was essential for the induction. The functional significance of the C/EBP site as indicated by the immunohistochemical result was that in HASMCs anti-C/EBPbeta reactivity was shifted from the cytoplasm to the nuclei. We have, therefore, demonstrated that the region containing the C/EBPalpha,beta consensus binding site between the bases -252 and -175 is important for the glucocorticoid-induced SAA1 gene expression in HASMCs but not in HepG2 cells.

Production of long-chain polyunsaturated fatty acids by monoxenic growth of labyrinthulids on oil-dispersed agar medium.

A novel method is proposed for the production of long-chain polyunsaturated fatty acids (LCPUFA) by labyrinthulids. The method comprises a monoxenic culture with Psychlobacter phenylpyruvicus, using agar medium in which oil was dispersed. Soybean oil (SBO) was selected as the optimum material for an oil-dispersed agar medium. The labyrinthulids showed three-dimensional growth and an anastomosing ectoplasmic network in the SBO-dispersed agar medium. The oil plate changed from an opaque culture to a more transparent culture, due to growth of the labyrinthulids. The optimum culture conditions were 25-30 degrees C, an initial pH of 6-10 and artificial seawater with a salt concentration of 50-100%. These conditions are close to those where these strains were isolated. The maximum LCPUFA production (0.59 g/l) and dry cell weight (4.93 g/l) was obtained using strain S3-2 (isolated from Ishigaki Island) with 1.5% SBO at 14 days. This value was about 30 times more than that using glucose instead of SBO. The method proposed is promising in terms of the production of LCPUFA from reproducible oils.

Various scoring systems evaluating histologic features of chronic hepatitis C treated with interferon.

Various scoring systems for chronic hepatitis have been proposed; however, there is no standard scoring system for studies of interferon (IFN) therapy in patients with chronic hepatitis C. The aims of this study were to determine the most useful system reflecting histologic changes in biopsy specimens from complete responders and predicting the efficacy of IFN therapy. Patients with chronic hepatitis C were administered IFN-alpha for 6 months. Forty-six patients were included in this study and categorized as complete responders (n = 15), partial responders (n = 24), and nonresponders (n = 7) according to viral and biochemical responses to the therapy. Biopsy specimens obtained from each patient before and after treatment were evaluated under 3 different systems: Histological Activity Index (HAI), modified HAI, and Scheuer classification. Complete responders showed considerable improvement in both grade and stage on the modified HAI and Scheuer classifications. On the HAI, a considerable improvement was observed in grade but not in stage. No significant change was observed in partial responders or nonresponders on any system. Prediction of complete response was not possible under any system, but the pretreatment score reflecting piecemeal necrosis on any 1 of the 3 classifications and the fibrosis score on Scheuer classification were predictors of nonresponse. The modified HAI system and Scheuer classification were amply useful in evaluating histologic changes in complete responders. Scores higher than 4 of the categories reflecting piecemeal necrosis on any system and fibrosis scores of 3 or 4 on Scheuer classification predicted nonresponse to IFN therapy.

Significance of serum matrix metalloproteinases and their inhibitors on the antifibrogenetic effect of interferon-alfa in chronic hepatitis C patients.

OBJECTIVE AND METHODS: The imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) is considered to be an important determination of deposition and breakdown of the extracellular matrix. To investigate the antifibrogenetic effect of interferon-alpha (IFN-alpha) treatment on factors regulating hepatic fibrosis, serum MMP-1, MMP-2, TIMP-1 and TIMP-2 levels were measured by the one-step sandwich enzyme immunoassay in 27 patients with chronic hepatitis C and compared with the histological status of the patients before and at the end of treatment. RESULTS: After 6 months of IFN-alpha treatment, the histological status of liver fibrosis showed improvement in 9 patients (IF group) and no change or a worsening in 18 patients (NIF group). Compared with pretreatment levels, in the IF group, IFN treatment caused a significant increase in the MMP-1/TIMP-1 ratio. In the NIF group, however, the MMP-1/TIMP-1 ratio tended towards a decrease; moreover, there was not only a significant increase in TIMP-2 levels but also a tendency towards an increase in TIMP-1 levels. CONCLUSION: These results suggested that an elevated MMP-1/TIMP-1 ratio may ameliorate liver fibrosis by interferon in cases of chronic hepatitis C, whereas elevated levels of TIMP-1 and TIMP-2 might impede improvement.

Clinical significance of the insulin resistance index as assessed by homeostasis model assessment.

To examine the clinical significance of the insulin resistance index as determined by homeostasis model assessment (HOMA-IR), we investigated the relationship between HOMA-IR and the insulin resistance estimated by the euglycemic-hyperinsulinemic clamp method in various subgroups and compared the significance of HOMA-IR with that of fasting plasma insulin levels (FIRI). HOMA-IR was significantly correlated to the inverse of the glucose infusion rate (1/GIR) in both diabetic and non-diabetic subjects (r=0.747, P<0.0001 and r=0.419, P<0.002, respectively). In the diabetic patients, treatment with sulfonylureas did not weaken this correlation (r=0.833, P<0.0001). HOMA-IR was found to be closely related to FIRI (r=0.932, P<0.0001), but HOMA-IR was more closely associated with 1/GIR than FIRI was. HOMA-IR as well as 1/GIR was correlated with the visceral fat area (VFA) more closely than with the subcutaneous fat area (SFA), while FIRI was correlated almost equally with both of them. In conclusion, HOMA-IR is a convenient and beneficial method for evaluating insulin resistance, especially in subjects with visceral fat accumulation, and reflects insulin resistance obtained by euglycemic clamp more accurately than FIRI alone.

Discrimination of brain abscess and cystic tumor by in vivo proton magnetic resonance spectroscopy.

Proton magnetic resonance (MR) spectroscopy was evaluated for the differentiation of brain abscesses and cystic brain tumors. Proton MR spectroscopy was performed in vivo in two patients with brain abscess and eight patients with various cystic brain tumors (anaplastic astrocytoma, glioblastoma, and metastatic brain tumor). MR imaging with contrast medium demonstrated ring-like enhanced mass lesions in all patients. The various resonance peaks in proton MR spectra were assigned to metabolites according to chemical shifts. Treatment of the cystic brain lesions was based on the information from proton MR spectroscopy. Aspirated pus from one patient with brain abscess was examined using ex vivo proton MR spectroscopy. The in vivo spectra of brain abscess contained resonance peaks attributed to acetate, lactate, alanine, amino acids, and lipids in both cases, and an additional peak of succinate in one case. In vivo spectra of the neoplasms contained resonance peaks corresponding to lactate, lipids, choline, creatine, and N-acetyl aspartate. Proton MR spectroscopy is useful for discriminating brain abscess from cystic tumors with similar neuroimaging appearance, which is very important for determining the treatment strategy.

Dexamethasone, but not IL-1 alone, upregulates acute-phase serum amyloid A gene expression and production by cultured human aortic smooth muscle cells.

Although the SAA1 and SAA2 protein isoforms (A-SAA) of the serum amyloid A (SAA) family of acute phase reactants have been found in a number of extrahepatic tissues; the site of synthesis of extrahepatic SAA remains to be clarified. To investigate site(s) of synthesis of the SAA protein localized to atherosclerotic plaque, expression of the SAA1 and SAA2 genes by cultured human aortic smooth muscle cells (HASMC) was investigated. A-SAA protein isoforms were detectable by immunoblot analysis in the culture medium of HASMC. Both A-SAA and C-SAA (SAA4) mRNA isoforms were constitutively expressed by HASMC, but not, however, by the human umbilical vein endothelial cells. Expression of A-SAA mRNA by HASMC was upregulated by corticoid hormones including dexamethasone (Dex), corticosterone, hydrocortisone, and aldosterone, but not by the cytokines interleukin (IL)-1, IL-6, and tumour necrosis factor (TNF)-alpha alone. Dex stimulation of A-SAA mRNA was time and dose dependent from 6 to 48 h. The threshold concentration for upregulation of A-SAA mRNA in HASMC by Dex was between 0.1 and 1 nM. IL-1, known to upregulate extrahepatic A-SAA gene expression in other cell systems only slightly, if at all, upregulated Dex-induced A-SAA expression by HASMC. Thus, it is possible that some of the A-SAA protein in the vascular wall (atherosclerotic plaques) can originate from smooth muscle cells. In consideration of recent reports that A-SAA modulates the inflammatory process and lipid synthesis, A-SAA can potentially serve as a physiological regulator of smooth muscle cell homeostasis within that, in a disease state, participates in the formation of atherosclerotic plaques.

A polymorphism in the promoter region of the glucocorticoid receptor gene is associated with its transcriptional activity.

Since glucocorticoid exerts its biological effects by binding to its receptor, the expression efficiency of the glucocorticoid receptor (GR) gene could influence glucocorticoid sensitivity. We found a polymorphism of cytosine/adenine (-22 C/A) in the upstream region of the GR gene. There was no difference in the allelic frequency between normal and type 2 diabetic subjects. The promoter activity determined by luciferase assay was significantly lower in the -22 A allele than in the -22 C allele in both HepG2 (A allele, 4.19 +/- 0.15; C allele, 6.07 +/- 0.27, p < 0.001) and human embryonic kidney 293 cell lines (A allele, 0.93 +/- 0.16; C allele, 1.51 +/- 0.32, p < 0.001). This polymorphism is associated with transcription of the CR gene, which could be related to glucocorticoid sensitivity through an alteration in tissue GR number.

Serum arylesterase/diazoxonase activity and genetic polymorphisms in patients with type 2 diabetes.

Human serum paraoxonase (PON1) is associated with high-density lipoprotein (HDL) and inhibits the oxidation of low-density lipoprotein (LDL) in vitro, suggesting that PON1 protects against atherosclerosis. We detected 3 polymorphisms of the PON1 gene and investigated PON1 enzyme activities as paraoxonase (PON), arylesterase (ARYL) and diazoxonase (DIAZ), and serum PON1 concentration in 106 patients with type 2 diabetes and 161 control subjects. All 3 enzyme activities and specific activities of PON1 in diabetic patients were significantly lower than those in controls, while there was no difference in serum PON1 concentration between the patient and control groups. The specific activities of PON, ARYL, and DIAZ in patients were 6.82 +/- 3.14 nmol x min(-1) x U(-1) (mean +/- SD, U; unit for serum PON1 concentration), 4.77 +/- 0.17 micromol x min(-1) x U(-1), and 193 +/- 92 nmol x min(-1) x U(-1), respectively, whereas those in controls were 9.33 +/- 3.92 nmol x min(-1) x U(-1), 5.36 +/- 0.14 micromol x min(-1) x U(-1), and 242 +/- 103 nmol x min(-1) x U(-1), respectively. There was no significant difference in the allelic frequencies of -108C/T, 55L/M, or 192Q/R between the patient and control groups. When each enzyme activity was compared between the patient and control groups in each genotype subgroup, all activities were lower in the patient group. The PON and ARYL activities were lower in patients with retinopathy or nephropathy than in those without such complications, and the ARYL activity was also lower in patients with neuropathy. In conclusion, all specific enzyme activities of PON1 were lower in patients with type 2 diabetes independent of the -108C/T, 55L/M, or 192Q/R polymorphism, and this impaired PON1 function may be involved in development of diabetic microangiopathy.

Symbiobacterium thermophilum gen. nov., sp. nov., a symbiotic thermophile that depends on co-culture with a Bacillus strain for growth.

A Gram-negative and tryptophanase-positive thermophile, whose growth is dependent on co-culture with an associating Bacillus strain, had been reported and tentatively named Symbiobacterium thermophilum strain T(T). Axenic culture of strain T(T) was recently established by dialysing cultures with the supporting bacterial strains or adding their culture broth. Phylogenetic analysis of strain T(T), based on the 16S rDNA sequence, was conducted for the validation of S. thermophilum. The sequence of strain T(T) was located at the outermost position in the high-G+C Gram-positive group distinctly isolated from any other branches hitherto known. Ten sequences identical to that of strain T(T), and one sequence closely related to it, were identified for the first time from soil and compost samples. The outer membrane of strain T(T) had a three-layered structure, outside the cytoplasmic membrane, which is similar to the S-layer in the cells of members of the Bacillaceae. Chemical analysis of the cells revealed that menaquinone-6 is a major component of the quinone system. According to these results, along with several previous observations (i.e. a G+C DNA content of 65 mol% and the identification of iso-C15:0 and iso-C17:0 acids as major cellular fatty acids), the new taxon Symbiobacterium thermophilum gen. nov., sp. nov. is proposed. The type strain is S. thermophilum strain T(T) (= IAM 14863T).

A polymorphism upstream from the human paraoxonase (PON1) gene and its association with PON1 expression.

Human serum paraoxonase (PON1) is an esterase that has been shown to decrease the susceptibility of lipoproteins to lipid peroxidation. We found a polymorphism of cytosine/thymidine (-108C/T, the number is from the ATG codon) in the upstream region of the PON1 gene. The luciferase activity was lower in the -108T allele than in the -108C allele. The serum PON1 concentrations in 132 normal subjects were as follows: -108CC>-108CT and>-108TT genotypes. The polymorphism upstream from the PON1 gene is associated with transcription of the PON1 gene and the serum PON1 concentration.

Histochemical cytochrome c oxidase activity and caspase-3 in gerbil hippocampal CA1 neurons after transient forebrain ischemia.

We examined the cytochrome c oxidase (COX) activity in gerbil hippocampal CA1 neurons after 5-min ischemia by a histochemical method in the presence or absence of exogenous cytochrome c. In the CA1 neurons, COX activity without exogenous cytochrome c decreased from 1 h after ischemia, but was restored by the addition of exogenous cytochrome c in the following 6 h after ischemia. These results suggest that it is not COX activity but endogenous cytochrome c that is changed in the early phase after ischemia, and that COX activity begins to decrease 9 h after ischemia. We examined caspase-3 in the CA1 region by immunoblotting, as caspase-3 is known to take part in the cell-death cascade downstream from cytochrome c. Although pro-caspase-3 was strongly detected, active caspase-3 was not detected before and until 84 h after 5-min ischemia. Our data suggested that delayed neuronal death is likely to progress via cytochrome c-release but not via caspase-3 activation.