Plasminogen activator inhibitor and the risk of cardiovascular disease: The Framingham Heart Study.

INTRODUCTION: Although plasminogen activator inhibitor (PAI-1) plays a key regulatory role in fibrinolysis, it has not been clearly shown to independently predict cardiovascular disease (CVD) among individuals without prior CVD. We investigated, in the Framingham Heart Study offspring cohort, whether PAI-1 predicted CVD risk among individuals without prior CVD. METHODS: Plasma PAI-1 antigen and tissue plasminogen activator (TPA) antigen were measured in 3203 subjects without prior CVD between 1991 and 1995; average follow-up of 10 years. PAI-1 was remeasured 4 years after baseline, to determine the effect of serial change on risk. RESULTS: PAI-1 levels (mean ± SD) were 29.1 ng/ml (19.2) versus 22.1 (16.5) for those and without incident CVD; p<0.001, and TPA levels were 12.0 ng/ml (5.7) versus 9.0 (4.7); p<0.001. PAI-1 and TPA antigen levels had a strong unadjusted linear relation with incident CVD (p<0.001). After adjustment for conventional risk factors, the hazard ratios (HRs) for higher quartiles of PAI-1, compared with the lowest, were 1.9, 1.9, 2.6 (linear trend p=0.006), and 1.6, 1.6, 2.9 (p<0.001) for TPA antigen. The adjusted HRs for increasing quartiles of serial change in PAI-1 at 4 years, compared with the lowest, were 0.9, 0.8, 1.3 (p=0.050). C statistic assessment showed that adding PAI-1 or TPA to conventional risk factors resulted in small increases in discrimination and modest reclassification of risk, which was statistically significant for TPA (net reclassification 6.8%, p=0.037) but not PAI-1 (4.8%, p=0.113). CONCLUSION: PAI-1 and TPA antigen levels are predictive of CVD events after accounting for established risk factors. A serial increase in PAI-1 is associated with a further increase in risk. These findings support the importance of fibrinolytic potential in CVD.

Circulating microRNAs predict biochemical recurrence in prostate cancer patients.

BACKGROUND: Circulating microRNAs (miRNAs) are emerging as promising biomarkers for prostate cancer. Here, we investigated the potential of these molecules to assist in prognosis and treatment decision-making. METHODS: MicroRNAs in the serum of patients who had experienced rapid biochemical recurrence (BCR) (n=8) or no recurrence (n=8) following radical prostatectomy (RP) were profiled using high-throughput qRT-PCR. Recurrence-associated miRNAs were subsequently quantitated by qRT-PCR in a validation cohort comprised of 70 patients with Gleason 7 cancers treated by RP, 31 of whom had undergone disease progression following surgery. The expression of recurrence-associated miRNAs was also examined in tumour tissue cohorts. RESULTS: Three miRNAs - miR-141, miR-146b-3p and miR-194 - were elevated in patients who subsequently experienced BCR in the screening study. MiR-146b-3p and miR-194 were also associated with disease progression in the validation cohort, as determined by log-rank tests and Cox proportional hazards regression. Multivariate analysis revealed that miR-146b-3p possessed prognostic information beyond standard clinicopathological parameters. Analysis of tissue cohorts revealed that miR-194 was robustly expressed in the prostate, elevated in metastases, and its expression in primary tumours was associated with a poor prognosis. CONCLUSION: Our study suggests that circulating miRNAs, measured at the time of RP, could be combined with current prognostic tools to predict future disease progression in men with intermediate risk prostate cancers.

Outcome of post-prostatectomy radiotherapy in one institution.

We present a retrospective study to evaluate the outcome of postoperative radiotherapy for biochemical or clinical recurrent prostate cancer. Twenty-six patients (median age 60 years) underwent radiotherapy after radical prostatectomy between January 1997 and January 2004. Seven patients received adjuvant radiotherapy and 19 received salvage radiotherapy. The median prostate-specific antigen at diagnosis was 8.6 (0.9-89) and most (23 patients) presented with T(3)N(0) disease. The median follow up was 19.5 months (5-84 months). All patients received a dose of 61.2 Gy at 1.8 Gy per fraction, 20 initially receiving 45 Gy to the lesser pelvis. The median dose to the bladder, rectum and left femoral head were 55.6, 57.5 and 33.8 Gy, respectively. All patients were managed radiotherapeutically by the first author. Twenty-four patients are alive. Two patients have died, one from oesophageal cancer and the second from metastatic prostate cancer. Two other patients also developed metastatic disease. Four asymptomatic patients with a rising prostate-specific antigen are under observation. None of the 26 patients has developed a local recurrence. Seven patients have developed grade 1 late bowel effects and three a grade 2 late effect. Eight patients suffer from grade 1 late genitourinary effects and two from grade 2 effects. One patient developed impotence, whereas 23 patients were rendered impotent postoperatively. There were no grade 3/4 late effects. Postoperative radiotherapy is well tolerated and provides effective local control.

A 6-month study of the efficacy and safety of tadalafil in the treatment of erectile dysfunction: a randomised, double-blind, parallel-group, placebo-controlled study in Australian men.

The efficacy and safety of tadalafil for the treatment of erectile dysfunction (ED) were assessed in a 6-month, randomised, double-blind, placebo-controlled study. Australian men with mild, moderate or severe ED of organic, psychogenic or mixed aetiology were randomised to tadalafil 20 mg as needed (n = 93) or placebo (n = 47). Efficacy assessments included the international index of erectile function (IIEF) and the sexual encounter profile (SEP) diary. Tadalafil significantly improved erectile function compared with placebo (p < 0.001, all measures). At the end of the study, the mean per-patient proportion of successful sexual intercourse attempts (SEP question three) was 73.5% for patients treated with tadalafil and 26.8% for placebo-treated patients. Improved erections were reported by 78% of tadalafil-treated patients compared to 12.8% of placebo-treated patients. The most common treatment-emergent adverse events--headache and dyspepsia--were generally mild or moderate. Tadalafil was effective and well tolerated in Australian men with mild to severe ED.

Cytochemistry and immunolocalisation of polysaccharides and proteoglycans in the endosperm of green Arabica coffee beans.

The major noncellulosic polysaccharides and proteoglycans in the coffee bean (Coffea arabica) cell wall are (galacto)mannans and arabinogalactan proteins. Immunological and chemical probes demonstrated that the mannans and arabinogalactan proteins were located continuously across the width of the cell wall, but that the concentration of different structural epitopes within these polysaccharide types showed considerable spatial variation. For the mannans this was implied by the striated pattern demonstrated by fluctuation of the affinity between the mannan monoclonal antibody BGM C6 and (galacto)mannan. The arabinogalactan proteins labelled by the Yariv reagent and the arabinogalactan protein-specific antibody LM2 appeared to be located in all regions of the wall except the middle lamella, but showed some differences in intensity of labelling. However, the LM6 antibody, specific for (1-->5)-alpha-arabinan epitopes, was located only as a compact region adjacent to the cell lumen in the body of the endosperm; though, it did label throughout the wall of epidermal cells. This implied that either some of the more highly arabinosylated arabinogalactan proteins contained contiguous 5-arabinosyl residues or that a rhamnogalacturonan which contained 5-arabinosyl residues as side chains existed in the cell wall. In either case the polymers were very restricted in their distribution. A second category of pectin, a homogalacturonan detected by JIM7, was located only in the middle lamella region. The architecture of the wall, as revealed by resin etching, appeared to reflect the chemical heterogeneity, with three distinct physical zones identifiable in a cross section across a single wall.

Sieve element specific labeling with an anti-idiotypic monoclonal antibody mimic of the phytotoxin dothistromin.

A monoclonal antibody, 12C9, an anti-idiotypic mimic of dothistromin, a toxin produced by Dothistroma pini, was found to label the cell wall of sieve elements in a number of different plant tissues and species. The antibody labeled apple leaf tissue, tobacco leaf mid vein, leaf and meristem, and Coprosma robusta leaf mid vein. Labeling was restricted to cell walls of sieve elements and did not label the companion cells or the lumen of the cells. The antibody labeled over a wide range of dilutions. This antibody could be used to differentiate sieve elements from other types of phloem. It could also be used to co-localize sieve elements and microorganisms such as phytoplasmas stained with DAPI.

Obstetric anaesthesia training in the Wessex region before and after introduction of Calman training.

We canvassed the opinions of anaesthetic trainees by questionnaire in 1995 and 1998, before and after the introduction of Calman training in which the registrar and senior registrar grades were replaced by the specialist registrar grade. We received replies from 106 trainees in 1995 (90%) and 115 (92%) in 1998. The survey results demonstrate that the total experience in obstetric anaesthesia gained by trainees has not decreased. Experience of regional techniques (epidural, spinal and combined spinal-epidural) increased, but the proportion of senior trainees who had performed fewer than 20 general anaesthetics for caesarean section rose from 0/23 in 1995 to 4/33 (12%). In 1998, the majority of senior trainees had experience of general anaesthesia for fetal distress, severe preeclampsia, eclampsia and massive obstetric haemorrhage. Only a minority had experienced failed intubation or a total spinal. In 1995, 5/21 (24%) of senior house officers agreed or strongly agreed that they were on call before they felt confident about dealing with common problems. The proportion was still 4/23 (17%) in 1998.

Ultrastructural changes to the midgut of the black field cricket (Teleogryllus commodus) following ingestion of potato protease inhibitor II.

Ultrastructural changes to the midgut epithelium of nymphs of the black field cricket (Teleogryllus commodus) after ingestion of potato protease inhibitor II (PPI-II) (0.6% (w/v) in artificial diet) were determined by light and electron microscopy. Crickets fed diet containing PPI-II grew more slowly than those fed control diet and changes observed to the PPI-II-fed nymphs included reduction of midgut wall depth, vacuolisation of the epithelial cells, swelling of the microvilli, cellular protrusions into the midgut and eventual rupture of individual or small groups of epithelial cells. These changes were first seen 2 days after PPI-II ingestion. Complete disintegration of the midgut to the basement membrane was not seen during the 27-day observation period and repair and regeneration of pockets of epithelial cells was observed. Immunocytochemistry revealed that PPI-II was localised within the ectoperitrophic matrix space of the gut. The location of the peritrophic matrix was determined by labelling with wheat germ agglutinin (WGA), but no rupture of this structure was observed in PPI-II-fed nymphs.

Alcohol consumption and hemostatic factors: analysis of the Framingham Offspring cohort.

BACKGROUND: Moderate alcohol consumers have lower rates of cardiovascular disease than abstainers. One proposed mechanism is a beneficial effect on hemostatic parameters, but previous studies have provided conflicting results. METHODS AND RESULTS: We measured levels of fibrinogen, plasma viscosity, von Willebrand factor, factor VII, plasminogen activator inhibitor antigen-1, and tissue plasminogen activator antigen in a cross-sectional analysis of 3223 adults free of cardiovascular disease enrolled in the Framingham Offspring Study. We assessed their alcohol consumption with a standardized questionnaire. Light-to-moderate alcohol consumption was associated with lower levels of fibrinogen, plasma viscosity, von Willebrand factor, and factor VII. This association was most pronounced for consumers of 3 to 7 drinks weekly for viscosity and 7 to 21 drinks weekly for the other hemostatic measures. Alcohol intake of 7 to 21 drinks weekly or more was associated with impaired fibrinolytic potential, reflected by higher levels of plasminogen activator inhibitor antigen-1 and tissue plasminogen activator antigen. Wine drinkers had lower plasminogen activator inhibitor antigen-1 levels than other drinkers, particularly at 3 to 21 drinks weekly, but beverage type did not otherwise consistently affect the results. CONCLUSIONS: Light-to-moderate alcohol consumption is associated with lower levels of coagulatory factors, but higher intake is associated with impaired fibrinolytic potential. These findings are consistent with the hypothesis that a balance between hemostatic and fibrinolytic activity may contribute to the complex relation of alcohol use with coronary heart disease.

Dark green islands in plant virus infection are the result of posttranscriptional gene silencing.

Dark green islands (DGIs) are a common symptom of plants systemically infected with a mosaic virus. DGIs are clusters of green leaf cells that are free of virus but surrounded by yellow, virus-infected tissue. We report here on two lines of evidence showing that DGIs are caused by posttranscriptional gene silencing (PTGS). First, transcripts of a transgene derived from the coat protein of Tamarillo mosaic potyvirus (TaMV) were reduced in DGIs relative to adjacent yellow tissues when the plants were infected with TaMV. Second, nontransgenic plants coinfected with TaMV and a heterologous virus vector carrying TaMV sequences showed reduced titers of the vector in DGIs compared with surrounding tissues. DGIs also were compared with recovered tissue at the top of transgenic plants because recovery has been shown previously to involve PTGS. Cytological analysis of the cells at the junction between recovered and infected tissue was undertaken. The interface between recovered and infected cells had very similar features to that surrounding DGIs. We conclude that DGIs and recovery are related phenomena, differing in their ability to amplify or transport the silencing signal.

Platelet glycoprotein IIIa Pl(a) polymorphism, fibrinogen, and platelet aggregability: The Framingham Heart Study.

BACKGROUND: Recent data suggest that the Pl(A2) allele of the platelet glycoprotein IIIa receptor may be a genetic risk factor for cardiovascular disease. We previously reported that the Pl(A2) allele was associated with increased platelet aggregability, as indicated by lower epinephrine threshold concentrations. Paradoxically, however, it has been reported that Pl(A2)-positive platelets have reduced fibrinogen binding. Because fibrinogen mediates platelet aggregability, we hypothesized that plasma fibrinogen levels may interact with Pl(A) genotype in modulating platelet aggregability. Methods and Results-- Glycoprotein IIIa Pl(A) genotype, fibrinogen level, and platelet aggregability were ascertained in 1340 subjects enrolled into the Framingham Offspring Study. Platelet aggregability was evaluated by the Born method. Higher fibrinogen levels were associated with increased epinephrine-induced aggregation (P=0.002) and a trend for ADP-induced aggregation (P=0.07). The fibrinogen effect was genotype specific, however, in that the increase in platelet aggregability with higher fibrinogen was present for the Pl(A1/A1) genotype (P=0.0005 and P=0.03 for epinephrine- and ADP-induced aggregation, respectively) but not for the Pl(A2)-positive genotype (P>0.90). CONCLUSION: Higher fibrinogen levels were associated with increased platelet aggregability. However, the association between fibrinogen and platelet aggregability was genotype specific. This interaction may be responsible for the conflicting findings regarding Pl(A) genotype and platelet aggregability. Further study of this gene-environment interaction may provide insight into cardiovascular disease risk.

Genetic and environmental contributions to platelet aggregation: the Framingham heart study.

BACKGROUND: Platelet aggregation plays an important role in arterial thrombosis in coronary heart disease, stroke, and peripheral arterial disease. However, the contribution of genetic versus environmental influences on interindividual variation in platelet aggregability is poorly characterized. METHODS AND RESULTS: We studied the heritability of platelet aggregation responses in 2413 participants in the Framingham Heart Study. The threshold concentrations of epinephrine and ADP required to produce biphasic platelet aggregation and collagen lag time were determined. Mixed-model linear regression was used to calculate correlation coefficients within sibships and within spouse pairs. Variance and covariance component methods were used to estimate the proportion of platelet aggregation attributable to measured covariates versus additive genetic effects. After accounting for environmental covariates, the adjusted sibling correlations for epinephrine, ADP, and collagen lag time were 0.24, 0.22, and 0.31, respectively (P=0.0001 for each). In contrast, adjusted correlations for spouse-pairs were -0.01, 0.05, and -0.02, respectively (all P>0.30). The estimated heritabilities were 0.48, 0.44, and 0.62, respectively. Measured covariates accounted for only 4% to 7% of the overall variance in platelet aggregation, and heritable factors accounted for 20% to 30%. The platelet glycoprotein IIIa Pl(A2) polymorphism and the fibrinogen Hind III beta-148 polymorphism contributed <1% to the overall variance. CONCLUSIONS: In our large, population-based sample, heritable factors play a major role in determining platelet aggregation, and measured covariates play a lesser role. Future studies are warranted to identify the key genetic variants that regulate platelet function and to lay the groundwork for rational pharmacogenetic approaches.

The use of thromboembolic deterrent stockings and a sequential compression device to prevent spinal hypotension during caesarean section.

Hypotension is a common side effect of spinal anaesthesia for caesarean section. We have performed a randomised, controlled study to determine the efficacy of a sequential compression device (SCD) (Kendall) in combination with thromboembolic deterrent (TED) stockings (Kendall) to reduce the incidence of hypotension in this setting. Within 20 min of spinal injection, there was no statistically significant difference in the incidence of hypotension (defined as less than 100 mmHg and less than 80% of baseline blood pressure) (TED/SCD group 65%, control 80%, P = 0.12). However, there was a trend for those receiving TED/SCD prophylaxis to require less ephedrine to maintain normotension than the control group (median TED/SCD 3 mg, control 6 mg, P = 0.08). The administration of ephedrine deviated from protocol on a total of 46 occasions (2.3% of recordings). To try to reduce the influence of this, we reinspected our data using time to first episode of hypotension with a Kaplan-Meier survival analysis. This showed that the instantaneous risk (hazard) of developing hypotension was 1.8 (95% CI: 1.1-2.9) times higher in controls than those receiving TED/SCD prophylaxis (P = 0.02). Despite demonstrating some benefit of TED/SCD prophylaxis to prevent hypotension, we do not consider that the magnitude of this benefit warrants their routine use.

Association of fibrinogen with cardiovascular risk factors and cardiovascular disease in the Framingham Offspring Population.

BACKGROUND: Fibrinogen has been identified as an independent risk factor for cardiovascular disease and associated with traditional cardiovascular risk factors. Also, the role of elevated fibrinogen in thrombosis suggests that it may be on the causal pathway for certain risk factors to exert their effect. These associations remain incompletely characterized. Moreover, the optimal fibrinogen assay for risk stratification is uncertain. METHODS AND RESULTS: In 2632 subjects from cycle 5 of the Framingham Offspring Population, fibrinogen levels were determined with a newly developed immunoprecipitation test (American Biogenetic Sciences) and the functional Clauss method. With the immunoprecipitation method, there were significant linear trends across fibrinogen tertiles (P:<0.001) for age, body mass index, smoking, diabetes mellitus, total cholesterol, HDL cholesterol, and triglycerides in men and women. The Clauss method had significant results (P:<0.030), except for triglycerides in men. Fibrinogen levels were higher for those with compared with those without cardiovascular disease. After covariate adjustment, fibrinogen remained significantly higher in those with cardiovascular disease with the use of the immunoprecipitation test (P:=0.035 and P:=0.018 for men and women, respectively) but not with the Clauss method. CONCLUSIONS: Fibrinogen was associated with traditional cardiovascular risk factors. Elevation of fibrinogen may provide a mechanism for risk factors to exert their effect. Also, fibrinogen levels were higher among subjects with cardiovascular disease compared with those without disease. The immunoprecipitation test showed a stronger association with cardiovascular disease than the Clauss method, suggesting that it may be a useful screening tool to identify individuals at increased thrombotic risk.

Cell-to-cell movement of potexviruses: evidence for a ribonucleoprotein complex involving the coat protein and first triple gene block protein.

The triple gene block proteins (TGBp1-3) and coat protein (CP) of potexviruses are required for cell-to-cell movement. Separate models have been proposed for intercellular movement of two of these viruses, transport of intact virions, or a ribonucleoprotein complex (RNP) comprising genomic RNA, TGBp1, and the CP. At issue therefore, is the form(s) in which RNA transport occurs and the roles of TGBp1-3 and the CP in movement. Evidence is presented that, based on microprojectile bombardment studies, TGBp1 and the CP, but not TGBp2 or TGBp3, are co-translocated between cells with viral RNA. In addition, cell-to-cell movement and encapsidation functions of the CP were shown to be separable, and the rate-limiting factor of potexvirus movement was shown not to be virion accumulation, but rather, the presence of TGBp1-3 and the CP in the infected cell. These findings are consistent with a common mode of transport for potexviruses, involving a non-virion RNP, and show that TGBp1 is the movement protein, whereas TGBp2 and TGBp3 are either involved in intracellular transport or interact with the cellular machinery/docking sites at the plasmodesmata.