Coated-platelet levels in patients with Type 1 and with Type 2 diabetes mellitus

Coated-platelet levels were quantified in 58 people with Type 1 diabetes, 90 with Type 2 diabetes, and 54 non-diabetic controls. In diabetes high coated-platelet levels were related to smoking and glucose control drugs, but not to glycaemia or other drugs. Prospective studies should evaluate coated-platelets and complications and drug effects.

Glycation does not alter LDL-induced secretion of tissue plasminogen activator and plasminogen activator inhibitor-1 from human aortic endothelial cells

Diabetes may induce both quantitative and qualitative changes in lipoproteins, especially low-density lipoprotein (LDL). Effects of LDL glycation on endothelial cell secretion of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) have not been fully elucidated. Human aortic endothelial cell (HAEC) tPA and PAI-1 production were determined after incubation with LDL (50 to 500 microg/mL protein, 24 h) from three sources: (1) nondiabetic LDL (N-LDL) modified in vitro to form six preparations: native, nonmodified (N); glycated (G); minimally oxidized (MO); minimally oxidized and glycated (MOG); heavily oxidized (HO); and heavily oxidized and glycated (HOG); (2) in vivo glycated and relatively nonglycated LDL subfractions from type 1 diabetic patients; (3) LDL from type 1 diabetic patients and matched controls, which was subfractionated using density gradient ultracentrifugation. In experiments using LDL modified in vitro, the rate of tPA release by HAECs incubated with N-LDL (83 +/- 4 ng/mg cell protein/24 h) did not differ significantly from those incubated with G-LDL (73 +/- 7), MO-LDL (74 +/- 13), or MOG-LDL (66 +/- 15) and was not influenced by LDL concentration. The rate of PAI-1 release was similar in HAECs incubated with N-LDL (5.7 +/- 0.6 mug/mg cell protein/24 h), G-LDL (5.7 +/- 0.7), MO-LDL (5.5 +/- 0.8), or MOG-LDL (5.7 +/- 0.9) and was not influenced by LDL concentration. In contrast, tPA release was significantly decreased in cells incubated with LDL (10 microg/mL) modified extensively by oxidation, and averaged 45.2 +/- 5.0 and 43.7 +/- 9.9 ng/mg/24 h for HO-LDL and HOG-LDL, respectively, and was further decreased with increasing concentrations of the heavily oxidized LDL preparations. PAI-1 release was not significantly decreased relative to N-LDL in cells incubated with low concentrations (5 to 50 microg/mL) of HO-LDL and HOG-LDL, but was decreased to 3.2 +/- 0.5 and 3.1 +/- 0.7 microg/mg/24 h for HO-LDL and HOG-LDL at 200 microg/mL, respectively. Results using in vivo glycated versus nonglycated LDL showed that tPA and PAI-1 release did not differ between subfractions. Release of tPA averaged 5.11 +/- 0.6 and 5.12 +/- 0.7 ng/mg/24 h, whereas release of PAI-1 averaged 666 +/- 27 ng/mg/24 h and 705 +/- 30 ng/mg/24 h for nonglycated and glycated LDL subfractions, respectively. Using LDL of different density subclasses, tPA and PAI-1 release in response to LDL from diabetic patients compared with control subjects did not differ when HAECs were incubated with LDLs of increasing density isolated from each subject pair. We conclude that oxidation of LDL, but not glycation, may contribute to the altered fibrinolysis observed in diabetes.

Platelet plasminogen activator inhibitor 1 in patients with type II diabetes

To compare platelet plasminogen activator inhibitor 1 (PAI-1) concentration in type II diabetic patients and healthy control subjects.

Stimulation of cholesteryl ester synthesis in human monocyte-derived macrophages by low-density lipoproteins from type 1 (insulin-dependent) diabetic patients:the influence of non-enzymatic glycosylation of low-density lipoproteins

Diabetes mellitus is an independent risk factor in the development of atherosclerosis. In this study we aimed to demonstrate whether there is an abnormal interaction between low-density lipoproteins from diabetic patients and human macrophages. We measured cholesteryl ester synthesis and cholesteryl ester accumulation in human monocyte-derived macrophages (obtained from non-diabetic donors) incubated with low density lipoproteins from Type 1 (insulin-dependent) diabetic patients in good or fair glycaemic control. Low density lipoproteins from the diabetic patients stimulated more cholesteryl ester synthesis than low density lipoproteins from non-diabetic control subjects (7.19 +/- 1.19 vs 6.11 +/- 0.94 nmol/mg cell protein/20 h, mean +/- SEM, p less than 0.05). The stimulation of cholesteryl ester synthesis by low density lipoproteins isolated from diabetic patients was paralleled by a significant increase in intracellular cholesteryl ester accumulation (p less than 0.02). There were no significant differences in the lipid composition of low density lipoproteins between the diabetic and control groups. Non-enzymatic glycosylation of low density lipoproteins was higher in the diabetic group (p less than 0.01) and correlated significantly with cholesteryl ester synthesis (r = 0.58). Similarly, low-density lipoproteins obtained from non-diabetic subjects and glycosylated in vitro stimulated more cholesteryl ester synthesis in macrophages than control low density lipoproteins. The increase in cholesteryl ester synthesis and accumulation by cells exposed to low density lipoproteins from diabetic patients seems to be mediated by an increased uptake of these lipoproteins by macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of interleukin-6 polymorphisms on human longevity:a systematic review and meta-analysis

Several studies have assessed changes in frequency of -174 interleukin (IL)-6 single nucleotide polymorphism (SNP) with age. If IL-6 tracks with disability and age-related diseases, then there should be reduction, in the oldest old, of the frequency of homozygous GG subjects, who produce higher IL-6 levels. However, discordant results have been obtained. To explore the relationship between this polymorphism and longevity, we analyzed individual data on long-living subjects and controls from eight case-control studies conducted in Europeans, using meta-analysis. There was no significant difference in the IL-6 genotype between the oldest old and controls (Odds Ratio [OR]=0.96; 95% C.I.: 0.77-1.20; p=0.71), but there was significant between-study heterogeneity (I2=55.5%). In a subgroup analyses when male centenarians from the three Italian studies were included, the frequency of the IL-6 -174 GG genotype was significantly lower than the other genotypes (OR=0.49; 95% C.I.: 0.31-0.80; p=0.004), with no evidence of heterogeneity (I2=0%). Our data supports a negative association between the GG genotype of IL-6 SNP and longevity in Italian centenarians, with males who carry the genotype being two times less likely to reach extreme old age compared with subjects carrying CC or CG genotypes. These findings were not replicated in other European groups suggesting a possible interaction between genetics, sex and environment in reaching longevity.

Genetic variation in the 6p22.3 gene DTNBP1, the human ortholog of the mouse dysbindin gene, is associated with schizophrenia

Prior evidence has supported the existence of multiple susceptibility genes for schizophrenia. Multipoint linkage analysis of the 270 Irish high-density pedigrees that we have studied, as well as results from several other samples, suggest that at least one such gene is located in region 6p24-21. In the present study, family-based association analysis of 36 simple sequence-length-polymorphism markers and of 17 SNP markers implicated two regions, separated by approximately 7 Mb. The first region, and the focus of this report, is 6p22.3. In this region, single-nucleotide polymorphisms within the 140-kb gene DTNBP1 (dystrobrevin-binding protein 1, or dysbindin) are strongly associated with schizophrenia. Uncorrected, empirical P values produced by the program TRANSMIT were significant (P

Identification of a high-risk haplotype for the dystrobrevin binding protein 1 (DTNBP1) gene in the Irish study of high-density schizophrenia families

A recent report showed significant associations between several SNPs in a previously unknown EST cluster with schizophrenia. (1). The cluster was identified as the human dystrobrevin binding protein 1 gene (DTNBP1) by sequence database comparisons and homology with mouse DTNBP1. (2). However, the linkage disequilibrium (LD) among the SNPs in DTNBP1 as well as the pattern of significant SNP-schizophrenia association was complex. This raised several questions such as the number of susceptibility alleles that may be involved and the size of the region where the actual disease mutation(s) could be located. To address these questions, we performed different single-marker tests on the 12 previously studied and 2 new SNPs in DTNBP1 that were re-scored using an improved procedure, and performed a variety of haplotype analyses. The sample consisted of 268 Irish multiplex families selected for high density of schizophrenia. Results suggested a simple structure where the LD in the target region could be explained by 6 haplotypes that together accounted for 96% of haplotype diversity in the whole sample. From these six, a single high-risk haplotype was identified that showed a significant association with schizophrenia and explained the pattern of significant findings in the analyses with individual markers. This haplotype was 30 kb long, had a large effect, could be measured with two tag SNPs only, had a frequency of 6% in our sample, seemed to be of relatively recent origin in evolutionary terms, and was equally distributed over Ireland. Implications of these findings for follow-up and replication studies are discussed.

Association between the 5q31.1 gene neurogenin1 and schizophrenia

Multiple lines of evidence suggest that schizophrenia results from aberrant neurodevelopment. The neurogenin1 gene (neurog1) consists of a single 1,666 bp exon that encodes a basic helix-loop-helix (bHLH) transcription factor that causes neuronal differentiation and induces cortical and glutamatergic differentiation programs. Because of its function and its location in 5q31.1, which has been linked to schizophrenia in multiple samples, we tested it for association with the disorder. We sequenced neurog1 in 25 affected subjects from the Irish Study of High-Density Schizophrenia Families. We observed a 5'-UTR SNP at position -60, already present in databases as rs8192558, and tested it along with rs2344485, rs8192559, and rs2344484. Narrow, intermediate, and broad diagnostic definitions were used. The major alleles of rs8192558 and rs2344484 were over-transmitted to affected subjects using both Pedigree Disequilibrium Test (PDT) (0.01 <or = P <or = 0.06) and FBAT (0.02 <or = P <or = 0.07). A haplotype consisting of the major alleles of all four SNPs was significantly over-transmitted in FBAT to the broad definition (P = 0.049), with trend significance to the narrow and intermediate definitions, and with trend significance in PDT. In confirmatory tests using 657 cases and 411 controls, this haplotype was slightly but not significantly over-represented in cases (81% vs. 77%, P = 0.21). These results, along with a priori evidence for the involvement of neurog1 in neurodevelopment, suggest that variants in neurog1 might have a small effect on susceptibility to schizophrenia. This gene should be tested in additional and larger samples.

Caveolin-1 single nucleotide polymorphism in antineutrophil cytoplasmic antibody associated vasculitis

Immunosuppression is cornerstone treatment of antineutrophil cytoplasmic antibody associated vasculitis (AAV) but is later complicated by infection, cancer, cardiovascular and chronic kidney disease. Caveolin-1 is an essential structural protein for small cell membrane invaginations known as caveolae. Its functional role has been associated with these complications. For the first time, caveolin-1 (CAV1) gene variation is studied in AAV.

Retroviral-mediated gene transfer of the human erythropoietin gene into a factor-dependent cell line, TF-1

The factor-dependent cell line, TF-1, established from a patient with erythroleukaemia, shows characteristics of immature erythroblasts. Addition of granulocyte-macrophage colony stimulating factor (GM-CSF) to the culture medium is required for long-term growth of the cells. Erythropoietin (Epo) can also be used to sustain TF-1 cells but for only limited periods (approximately a week). Low levels of both growth factors can act synergistically to maintain proliferation for a longer period of time than Epo alone. To eliminate the requirement of exogenous Epo for growth, TF-1 cells were co-cultured with a retroviral secreting cell line containing the human erythropoietin (hEpo) gene and a neomycin (neo) selectable marker. TF-1 cells which exhibited neo resistance (indicating infection by the retrovirus) were then grown in low concentrations of GM-CSF without the addition of Epo. Under these conditions growth of normal TF-1 cells was not sustained. The neo-resistant cells survived for more than 14 days indicating synergy between GM-CSF and the Epo synthesised by the co-cultured TF-1 cells. Radioimmunoassays performed on growth media detected concentrations up to 1 mU/ml of Epo, implying that stable integration of the retroviral vector and expression of the hEpo gene have been achieved.

Regulation of TGF-β1-Driven Differentiation of Human Lung Fibroblasts: Emerging Roles of Cathepsin B and Cystatin C

Lung matrix homeostasis partly depends on the fine regulation of proteolytic activities. We examined the expression of human cysteine cathepsins (Cats) and their relative contribution to TGF-β1-induced fibroblast differentiation into myofibroblasts. Assays were conducted using both primary fibroblasts obtained from patients with idiopathic pulmonary fibrosis (IPF) and human lung CCD-19Lu fibroblasts. Pharmacological inhibition and genetic silencing of Cat B diminished α-smooth muscle actin expression, delayed fibroblast differentiation and led to an accumulation of intracellular 50-kDa TGF-β1. Moreover addition of Cat B generated 25-kDa mature form of TGF-β1 in Cat B siRNA-pretreated lysates. Inhibition of Cat B decreased Smad 2/3 phosphorylation, but had no effect on p38 MAPK and JNK phosphorylation indicating that Cat B mostly disturbs TGF-β1-driven canonical Smad signaling pathway. While mRNA expression of cystatin C was stable, its secretion, which was inhibited by brefeldin A, increased during TGF-β1-induced differentiation of IPF and CCD-19Lu fibroblasts. In addition cystatin C participated in the control of extracellular Cats, since its gene silencing restored their proteolytic activities. These data support the notion that Cat B participates in lung myofibrogenesis as suggested for stellate cells during liver fibrosis. Moreover, we propose that TGF-β1 promotes fibrosis by driving the effective cystatin C-dependent inhibition of extracellular matrix-degrading Cats.

Expression of transketolase-like 1 protein (TKTL1) in human endometrial cancer

Malignant tumors metabolize glucose to lactate even in the presence of oxygen (aerobic glycolysis). The metabolic switch from oxidative glycolysis to non-oxidative fermentation of glucose and proteins performed by the tumor cells seems to be associated with TKTL1 and pAkt overexpression. Therefore the aim of the present study was to investigate the expression of TKTL1 and pAkt in human specimens of endometrial cancer as compared to benign endometrium. Additionally, expression of the glucose transporter GLUT1 was also investigated as aerobic glycolysis is associated with an increased need for glucose.

Two potential clinical applications of the alkaline single-cell gel electrophoresis assay .1. Human bladder washings and transitional cell carcinoma of the bladder .2. Human sperm and male infertility

Part 1: The alkaline single-cell gel electrophoresis (comet) assay was used to analyse the integrity and DNA content of exfoliated cells extracted from bladder washing specimens from 9 transitional cell carcinoma patients and 15 control patients. DNA damage, as expressed by % tail DNA and tail moment values, was observed to occur in cells from both control and bladder cancer samples. The extent of the damage was, however, found to be significantly greater in the cancer group than in the control group. Comet optical density values were also recorded for each cell analysed in the comet assay and although differences observed between tumour grades were not found to be statistically significant, the mean comet optical density value was observed to be greater in the cancer group than in the control population studied, These preliminary results suggest that the comet assay may have potential as a diagnostic tool and as a prognostic indicator in transitional cell carcinoma, Part 2: Baseline DNA damage in sperm cells from 13 normozoospermic fertile males, 17 normozoospermic infertile males and 11 asthenozoospermic infertile males were compared using a modified alkaline comet assay technique. No significant difference in the level of baseline DNA damage was observed between the 3 categories of sperm studied; however the untreated sperm cells were observed to display approximately 20% tail DNA. This is notably higher than the background DNA damage observed in somatic cells where the % tail DNA is normally less than 5%. Sperm from the 3 groups of men studied were also compared for sensitivity to DNA breakage, using the modified alkaline comet assay, following X-ray irradiations (5, 10 and 30 Gy) and hydrogen peroxide treatments (40, 100 and 200 mu M). Significant levels of X-ray-induced damage were found relative to untreated control sperm in the two infertile groups following 30 Gy irradiation. Significant damage in hydrogen peroxide-treated sperm was observed in sperm from fertile samples, at 200 mu M and in infertile samples at 100- and 200-mu M doses relative to controls. These results therefore indicate that fertile sperm samples are more resistant to X-ray- and hydrogen peroxide-induced DNA breakage than infertile samples. Further studies involving greater numbers of individuals are currently in progress to confirm these findings.

Metabolic signatures of human Alzheimer's disease (AD): H-1 NMR analysis of the polar metabolome of post-mortem brain tissue

Brain tissue from so-called Alzheimer's disease (AD) mouse models has previously been examined using H-1 NMR-metabolomics, but comparable information concerning human AD is negligible. Since no animal model recapitulates all the features of human AD we undertook the first H-1 NMR-metabolomics investigation of human AD brain tissue. Human post-mortem tissue from 15 AD subjects and 15 age-matched controls was prepared for analysis through a series of lyophilised, milling, extraction and randomisation steps and samples were analysed using H-1 NMR. Using partial least squares discriminant analysis, a model was built using data obtained from brain extracts. Analysis of brain extracts led to the elucidation of 24 metabolites. Significant elevations in brain alanine (15.4 %) and taurine (18.9 %) were observed in AD patients (p ≤ 0.05). Pathway topology analysis implicated either dysregulation of taurine and hypotaurine metabolism or alanine, aspartate and glutamate metabolism. Furthermore, screening of metabolites for AD biomarkers demonstrated that individual metabolites weakly discriminated cases of AD [receiver operating characteristic (ROC) AUC <0.67; p < 0.05]. However, paired metabolites ratios (e.g. alanine/carnitine) were more powerful discriminating tools (ROC AUC = 0.76; p < 0.01). This study further demonstrates the potential of metabolomics for elucidating the underlying biochemistry and to help identify AD in patients attending the memory clinic