Refractive index to evaluate staining specificity of extracellular vesicles by flow cytometry.

Extracellular vesicles (EVs) in plasma are commonly identified by staining with antibodies and generic dyes, but the specificity of antibodies and dyes to stain EVs is often unknown. Previously, we showed that platelet-depleted platelet concentrate contains two populations of particles >200 nm, one population with a refractive index (RI) < 1.42 that included the majority of EVs, and a second population with an RI > 1.42, which was thought to include lipoproteins. In this study, we investigated whether EVs can be distinguished from lipoproteins by the RI and whether the RI can be used to determine the specificity of antibodies and generic dyes used to stain plasma EVs. EVs and lipoproteins present in platelet-depleted platelet concentrate were separated by density gradient centrifugation. The density fractions were analyzed by Western blot and transmission electron microscopy, the RI of particles was determined by Flow-SR. The RI was used to evaluate the staining specificity of an antibody against platelet glycoprotein IIIa (CD61) and the commonly used generic dyes calcein AM, calcein violet, di-8-ANEPPS, and lactadherin in plasma. After density gradient centrifugation, EV-enriched fractions (1.12 to 1.07 g/mL) contained the highest concentration of particles with an RI < 1.42, and the lipoprotein-enriched fractions (1.04 to 1.03 g/mL) contained the highest concentration of particles with an RI > 1.42. Application of the RI showed that CD61-APC had the highest staining specificity for EVs, followed by lactadherin and calcein violet. Di-8-ANEPPS stained mainly lipoproteins and calcein AM stained neither lipoproteins nor EVs. Taken together, the RI can be used to distinguish EVs and lipoproteins, and thus allows evaluation of the specificity of antibodies and generic dyes to stain EVs.

Procoagulant tissue factor-exposing vesicles in human seminal fluid.

Recent studies indicate that various types of vesicles, like microparticles (MP) and exosomes, are present in blood, saliva, bone marrow, urine and synovial fluid. These vesicles, which are released upon activation or shear stress, are thought to play a role in coagulation, neovascularisation, inflammation and intercellular signalling. Seminal fluid is a cell-, sperm- and protein-rich suspension. Although seminal fluid is known to contain vesicles like prostasomes, MP and exosomes have never been characterised. Therefore, the aim of our study was to analyse and characterise vesicles in seminal fluid in male partners of patients undergoing controlled ovarian stimulation for IVF/ICSI. MP from seminal fluid of patients during routine IVF/ICSI procedures were detected and analysed with flow cytometry (FACS) and transmission electron microscopy (TEM), using antibodies against tissue factor (TF), CD10, CD13, CD26 and annexin V. The coagulant properties of vesicles were studied using a fibrin generation test. MP were detected in human seminal fluid by both flow cytometry and TEM. Seminal fluid-derived MP expressed CD10, CD13, CD26 and TF, which was highly procoagulant and a powerful trigger of the extrinsic pathway of coagulation. The extent to which the procoagulant activity of MP in seminal fluid contributes to the implantation process itself and therefore affects human reproduction needs to be further elucidated.

Active caspase-3 is removed from cells by release of caspase-3-enriched vesicles.

Cleavage of Rho associated Coiled Coil kinase I (ROCK I) by caspase-3 contributes to membrane blebbing. Whether caspase-3 and ROCK I also play a role in the release of membrane vesicles is unknown. Therefore, we transfected a human breast cancer cell line (MCF-7) that is caspase-3 deficient, lacks membrane blebbing, and does not release membrane vesicles, with caspase-3. Cells expressing caspase-3 demonstrate both ROCK I-mediated membrane blebbing, and release of small (400-600nm) membrane vesicles in a ROCK I-independent manner. These membrane vesicles contain caspase-3, and are enriched in caspase-3 activity compared to the releasing cells. Caspase-3-containing vesicles are taken up by untransfected cells but the cells do not show any sign of apoptosis. In conclusion, we show that the release of caspase-3-enriched membrane vesicles and membrane blebbing are two differentially regulated processes. Furthermore, we hypothesize that packaging of caspase-3 into membrane vesicles contributes to cellular homeostasis by the removal of caspase-3, and concurrently, protects the cells' environment from direct exposure to caspase-3 activity.

Cell-derived microparticles contain caspase 3 in vitro and in vivo.

BACKGROUND: Microparticles (MP) from endothelial cells (endothelial microparticles; EMP) circulate in disease states, but the processes such as apoptosis or cell activation underlying their release are unclear. OBJECTIVES: We investigated whether adherent (viable) or detached (apoptotic) endothelial cells are the possible source of EMP in vitro, i.e. under control and interleukin (IL)-1alpha activation conditions, and in vivo. METHODS: Adherent and detached endothelial cells, and EMP, were isolated from human umbilical vein endothelial cell cultures (n = 6), treated without or with IL-1alpha (5 ng mL(-1); 24 h). Cell fractions were analyzed by flow cytometry for annexin V binding, propidium iodide (PI) and caspase 3 staining (n = 3). Caspase 3 in EMP was studied using Western blot (n = 6) and flow cytometry (n = 6). Plasma from healthy subjects and systemic lupus erythematosus patients (both n = 3) were analyzed for caspase 3-containing (E)MP. RESULTS: Detached but not adherent cells double-stained for annexin V and PI, confirming the apoptotic conditions of the detached cells and the viable nature of the adherent cells. Caspase 3 was solely present in the detached cells and procaspase 3 in the adherent cells. Caspase 3 was present in EMP from both control and IL-1alpha-treated cultures. Counts of EMP and detached cells, but not adherent cells, highly correlated (r = 0.959, P < 0.0001). In vivo circulating MP from nucleated (endothelial cells, monocytes) and anucleated cells (platelets, erythrocytes) contained caspase 3. CONCLUSIONS: EMP contain caspase 3 and may be mainly derived from detached (apoptotic) endothelial cells in vitro. The presence of caspase 3 in MP from anucleated cell types, however, suggests that its presence may not necessarily be related to apoptosis in vivo but may be associated with caspase 3 activation unrelated to apoptosis.

Fiber intake, constipation, and risk of varicose veins in the general population: Edinburgh Vein Study.

The purpose of the present study was to determine the relationship between fiber intake, constipation, and clinical venous disease in the general population. The Edinburgh Vein Study was comprised of 1566 men and women aged 18-64 years who were selected at random from the age-sex registers of 12 general practices. Fiber intake, intestinal transit time, defecation frequency and the prevalence of straining at stool were all found to be significantly different between the sexes. Men who reported that they strained to start passing a motion showed a higher prevalence of mild and severe trunk varices compared to men who did not strain. After adjustment for social class, BMI and mobility at work, this group of men showed a significantly elevated risk of having severe trunk varices (OR 2.76, 95% CI 1.16, 6.58). In contrast, no consistent relationships were seen among women. Overall, within this Western general population, an association between dietary fiber, constipation and the presence or severity of varicose veins was not supported.

Tissue plasminogen activator and leucocyte elastase as predictors of cardiovascular events in subjects with angina pectoris: Edinburgh Artery Study.

AIMS: To evaluate the relationship between haemostatic and rheological factors and cardiovascular outcome in subjects with angina pectoris in the general population. METHODS AND RESULTS: Two hundred and seven men and women aged 55-74 had evidence of angina at baseline. Sixty-seven (32.3%) had a fatal or non-fatal cardiovascular event during follow-up. Median levels of tissue plasminogen activator antigen and leucocyte elastase were higher in the event group compared with the no event group (10.0 ng. ml(-1)vs 7.2 ng. ml(-1);P

Methylene tetrahydrofolate reductase (MTHFR) and nitric oxide synthase (ecNOS) genes and risks of peripheral arterial disease and coronary heart disease: Edinburgh Artery Study.

Hyperhomocysteinaemia and reduced nitric oxide synthesis may each result in endothelial dysfunction predisposing to atherogenesis. Genetic variants of methylene tetrahydrofolate reductase (MTHFR) and endothelial nitric oxide synthase (ecNOS) influence homocysteine metabolism and nitric oxide synthesis, respectively and might thus be determinants of the risk of atherosclerotic disease. The aim of our study was to identify, in a general population sample, the risks of peripheral arterial disease and of coronary heart disease related to MTHFR (175;198) and ecNOS (4;5) polymorphisms. In the Edinburgh Artery Study, which is a population based cohort study, 940 men and women aged 60-79 years, who had previously been selected at random from the general population, had DNA extracted from a venous blood sample. Based on a clinical examination at baseline and follow up investigations, three groups of subjects were identified: those with peripheral arterial disease (n=80), those with coronary heart disease (n=137), and healthy controls who had no evidence of cardiovascular disease (n=300). The distributions of the ecNOS and MTHFR genotypes did not differ significantly between the groups with and without cardiovascular disease. However, the ecNOS-4 allele (frequency 0.13) was related to the occurrence of coronary heart disease in non smokers, OR=2.47 (95% CI [1.42, 4.34], P=0.02). No association was found with peripheral arterial disease. The MTHFR-175 allele (frequency 0.31) was not related to coronary heart disease, but was associated with a reduced risk of peripheral arterial disease, OR=0.54 (95% CI [0.32, 0.90], P=0.02). Neither the ecNOS-4 allele or MTHFR-175 allele was related to the ankle brachial pressure index in the whole study population. In conclusion, the ecNOS-4 allele was associated with a slightly increased risk of coronary heart disease in non-smokers, but otherwise the MTHFR and ecNOS genotypes appeared to have little influence on the risks of peripheral arterial disease and coronary heart disease in this older population.

Plasma fibrinogen, haemostatic factors and prediction of peripheral arterial disease in the Edinburgh Artery Study.

The role of fibrinogen and other haemostatic factors in prediction of peripheral arterial disease (PAD) has not been established. We examined the associations of plasma fibrinogen, von Willebrand Factor (vWF), tissue plasminogen activator (t-PA) antigen, fibrin D-dimer, and factor VII with the development and clinical progression of PAD. In the Edinburgh Artery Study, 1592 men and women, aged between 55 and 74 years, were followed prospectively over 5 years to detect the onset of PAD, and the deterioration of established PAD. At baseline, 418 individuals had evidence of PAD and 60 (14.4%) subsequently deteriorated. 1080 subjects had no baseline disease, but 59 (5.5%) developed PAD during follow-up. Median levels of fibrinogen and vWF were higher in the group developing disease compared with the group which did not (2.78 g/l versus 2.57 g/l, P< or =0.01; 116 IU/dl versus 104 IU/dl, P< or =0.05; respectively). After adjusting for age and sex, fibrinogen (P< or =0.01) and vWF (P< or =0.05) were significantly associated with the risk of developing PAD. The association between fibrinogen and development of disease remained after adjusting for cardiovascular risk factors and baseline ischaemic heart disease (relative risk, 1.35, 95% confidence interval, 1.05, 1.73; P< or =0.05). None of the haemostatic factors were significantly associated with progression of PAD. In conclusion, plasma fibrinogen levels are related to the future onset of PAD, providing further evidence of a possible role of elevated fibrinogen in the development of atherosclerotic disease.

The role of haematological factors in diabetic peripheral arterial disease: the Edinburgh artery study.

The relationship between haematological factors and peripheral arterial disease (PAD) among diabetics has not been widely examined. 1592 men and women aged 55-74 years were selected from the general population. They underwent an assessment for PAD and a glucose tolerance test. 288 subjects (18.7%) were identified as having diabetes or impaired glucose tolerance (IGT). Among the diabetes/IGT group, median levels of fibrinogen, von Willebrand factor (VWF), tissue plasminogen activator (t-PA), fibrin D-dimer and plasma viscosity were higher in subjects with PAD than those without PAD (P

Role of systolic blood pressure and plasma triglycerides in diabetic peripheral arterial disease. The Edinburgh Artery Study.

OBJECTIVE: To determine the risk factors for peripheral arterial disease (PAD) in a diabetic population and to examine whether different levels of these risk factors might explain why diabetic subjects have an increased risk of PAD compared with normal glucose tolerance subjects. RESEARCH DESIGN AND METHODS: There were 1,592 men and women aged 55-74 years selected at random from the age-sex registers of 11 general practices in Edinburgh, Scotland. Subjects underwent a comprehensive medical examination, including assessment for PAD (intermittent claudication on World Health Organization questionnaire or major asymptomatic disease on noninvasive testing) and a glucose tolerance test. RESULTS: Of the subjects, 288 (18.7%) were found to have diabetes or impaired glucose tolerance (IGT). The prevalence of PAD was greater in those with diabetes/IGT (20.6%) compared with those with normal glucose tolerance (12.5%) (odds ratio [OR] 1.64, 95% CI 1.17-2.31). Among the diabetes/IGT group, mean levels of smoking, systolic blood pressure, and triglycerides were higher in subjects with PAD than in those without PAD (P < or = 0.05). Mean levels of systolic blood pressure and plasma triglycerides were also higher in diabetic subjects than in nondiabetic subjects with PAD (P < or = 0.05). In multivariate analysis, those with diabetes/IGT no longer had a significantly higher risk of PAD after adjusting separately for systolic blood pressure (OR 1.22, 95% CI 0.85-1.73) and plasma triglycerides (OR 1.26, 95% CI 0.89-1.79). Simultaneous adjustment for both systolic blood pressure and triglycerides reduced the risk of PAD among diabetic subjects to 1.11 (95% CI 0.78-1.58). CONCLUSIONS: Increased mean levels of triglycerides and systolic blood pressure may help to explain the higher prevalence of PAD in diabetic subjects compared with that in normal glucose tolerance subjects.

Molecular cloning of human gastric mucin MUC5AC reveals conserved cysteine-rich D-domains and a putative leucine zipper motif.

To further clone the human gastric mucin MUC5AC cDNA, we screened a human gastric cDNA library with previously identified MUC5AC sequences. We obtained 32 independent clones encoding newly identified sequences comprising the entire N-terminal sequence of MUC5AC, up to 3024 bp upstream of the previously identified MUC5AC sequences. The N-terminus of MUC5AC shows high homology (43% identity) with the N-terminus of MUC2 and contains three domains homologous to the D-domains found in the pro-von Willebrand factor. Furthermore, the N-terminus of MUC5AC contains a putative leucine zipper motif not found in any other mucin identified so far. Moreover, a large central repetitive sequence was identified encoding approximately 2500 amino acids (7.5 kb). We were able to establish that the MUC5AC cDNA together with the previously identified 6.1 kb of MUC5AC cDNA sequence is about 16.6 kb, encoding 5525 amino acids. A model of the domain structure of MUC5AC is presented.

Large-scale ambulatory study of postprandial jejunal motility in irritable bowel syndrome.

BACKGROUND: The relationship of small-bowel dysmotility to dietary intake in irritable bowel syndrome (IBS) is obscure. METHODS: This study evaluated postprandial jejunal motility in IBS patients classified as constipation-predominant (n = 25) or diarrhoea-predominant (n = 35) and compared results against 18 volunteers. Twenty-four-hour ambulatory jejunal manometry was carried out in all subjects, and recordings were analysed by microcomputer and visual assessment. RESULTS: By means of analysis of variance (fitting factors for channels, meals, and time periods) postprandial contraction frequency was greater in both patient groups compared with normal (constipation-predominant versus normal, diarrhoea-predominant versus normal; P < 0.001). In the constipation-predominant cohort, contraction amplitudes were lower (constipation-predominant versus normal; P < 0.002). Discrete cluster contractions occurred with similar frequency and duration in both patient and volunteer groups. CONCLUSIONS: Quantitative differences of postprandial jejunal contraction characteristics have been shown between patients with IBS and healthy volunteers. Contraction frequency is greater than normal in both diarrhoea- and constipation-predominant categories, whereas contraction amplitudes are lower in constipation-predominant patients.

A biostatistical study into the efficiency of individualism using nonisotopic chemiluminescent-enhanced NICE multilocus DNA probes.

The efficiency of individualisation using nonisotopic chemiluminescent- enhanced probes (NICE) was investigated by analysing DNA fingerprints obtained from 190 unrelated Caucasians. Novel analysis of the scoring procedure enabled us to include the comparison of 585 pairs of samples for each of two probes. When the results of NICE probes 33.6 and 33.15 were combined, the mean percentage band share between two unrelated individuals was 16.8% and the mean number of bands identified in an individual DNA fingerprint was 54.8. Results were compared with those obtained using isotopically labelled probes and suggest that the two labelling systems gave similar efficiencies for differentiating between individuals. Analysis of DNA fingerprints from 37 family trios (mother, child and father groups) gave a mutation rate of 0.10% when using NICE probes. The two labelling systems compared were equally efficient in establishing family relationships.

Separating the environmental and genetic factors that may be causes of bovine spongiform encephalopathy.

The initial cause of the bovine spongiform encephalopathy (BSE) epidemic is generally accepted to have been the feeding of infected animal protein to cattle. The proportion of animals affected in any year in a particular herd has generally been low. This suggests either considerable variation in the extent of challenge of the individual animals or variation in their susceptibility to challenge or both. There is known to be genetic variation in susceptibility in other spongiform encephalopathies, such as scrapie in sheep. However, earlier indications that there may be associations between the incidence of BSE in cattle and polymorphisms and mutations in the PrP gene have not been confirmed (Hunter et al. 1994). Here, we attempt to model the likely extent of challenge of the individual animals in five Holstein Friesian pedigree herds and also the distribution of incubation times to the date of clinical onset. By studying the incidence of the disease in related animals we first found that single locus genetic models fitted the data much better than a non-genetic model. This was the first statistical evidence found of genetic variation in susceptibility to BSE. A check on the model in which individual animals were randomly allocated to 'parents' showed that the result was due to the lack of allowance in the non-genetic model for those animals insufficiently challenged or, for non-genetic reasons, resistant to their level of challenge. Thus there is still no evidence, molecular or statistical, for genetic variation in susceptibility. The importance of checking the attribution of genetic effects in complex models by the random allocation of progeny to parents is clear.

The incidence of bovine spongiform encephalopathy in the progeny of affected sires and dams.

Case control study techniques were used to compare the incidence of bovine spongiform encephalopathy (BSE) in the progeny of two affected sires and 110 affected dams with the incidence of BSE in the progeny of animals known to be unaffected at the last record. All the progeny were born before the ban on ruminant-derived protein in feedstuffs issued in July 1988. The results provide little, if any, evidence of differences between the incidence in the progeny of the affected animals and the incidence in the progeny of the presumed unaffected animals. Data from five herds were used in a logistic regression analysis to study the effects of the disease status of the dam and the age of the dam at the birth of the calf on the incidence of BSE. The disease status of the dam did not significantly affect the disease status of its progeny, after allowance had been made for the effects of herd, year and the age at last record of the progeny. The difficulty of establishing maternal transmission if a high proportion of the dams are incubating the disease and transmission can occur early in the incubation period is discussed.

Clinical significance of fetal choroid plexus cysts.

Choroid plexus (CP) cysts are commonly detected on routine mid-trimester ultrasound scan. When associated anomalies are detected, the risk is sufficient to justify an invasive diagnostic test such as amniocentesis. However, the risk when no associated anomalies are detected is much less well defined. This information is required to determine the appropriate management in cases of apparently isolated CP cysts. We thought the only way to resolve the difficulties in counselling prospective parents was to conduct a prospective study in a large unselected population. A registry of fetal CP cysts detected over 3 years in the Yorkshire Region was compiled and we identified 524 CP cysts. These cases were then amalgamated and analysed with 1361 cases from prospective studies reported in the world English literature and a further 71 unpublished cases identified from a 2 year prospective series from Ninewells Hospital, Dundee. The risk of chromosomal abnormalities was 1 in 150 (95% CI 1 in 85, 1 in 261) when no fetal anatomic abnormalities, apart from the CP cysts themselves, were detected antenatally. The risk increased to approximately 1 in 3 if any other associated ultrasound abnormalities were detected antenatally. The risk did not appear to be related to whether or not cyst size diminished as gestation progresses, whether they were unilateral or bilateral, and whether they were small or large in size (60-80% < 10 mm). 76% of aneuploidic cases were trisomy 18 and 17% were trisomy 21. The risk of Down's syndrome in fetuses with CP cysts but no other anomalies detected antenatally is 1 in 880. The probability of a chromosomal abnormality is high when CP cysts are associated with any other antenatally detected anomaly, indicating a clear need to offering amniocentesis. The predictive value is much lower when no other anomalies are detected. In such cases, it is probably advisable to regard CP cysts as an indication for detailed ultrasound assessment, rather than invasive testing.