Bridging the age gap in breast cancer: impact of omission of breast cancer surgery in older women with oestrogen receptor-positive early breast cancer on quality-of-life outcomes.

BACKGROUND: Primary endocrine therapy may be an alternative treatment for less fit women with oestrogen receptor (ER)-positive breast cancer. This study compared quality-of-life (QoL) outcomes in older women treated with surgery or primary endocrine therapy. METHODS: This was a multicentre, prospective, observational cohort study of surgery or primary endocrine therapy in women aged over 70 years with operable breast cancer. QoL was assessed using European Organisation for Research and Treatment of cancer QoL questionnaires QLQ-C30, -BR23, and -ELD14, and the EuroQol Five Dimensions 5L score at baseline, 6 weeks, and 6, 12, 18, and 24 months. Propensity score matching was used to adjust for baseline variation in health, fitness, and tumour stage. RESULTS: The study recruited 3416 women (median age 77 (range 69-102) years) from 56 breast units. Of these, 2979 (87.2 per cent) had ER-positive breast cancer; 2354 women had surgery and 500 received primary endocrine therapy (125 were excluded from analysis due to inadequate data or non-standard therapy). Median follow-up was 52 months. The primary endocrine therapy group was older and less fit. Baseline QoL differed between the groups; the mean(s.d.) QLQ-C30 global health status score was 66.2(21.1) in patients who received primary endocrine therapy versus 77.1(17.8) among those who had surgery plus endocrine therapy. In the unmatched analysis, changes in QoL between 6 weeks and baseline were noted in several domains, but by 24 months most scores had returned to baseline levels. In the matched analysis, major surgery (mastectomy or axillary clearance) had a more pronounced adverse impact than primary endocrine therapy in several domains. CONCLUSION: Adverse effects on QoL are seen in the first few months after surgery, but by 24 months these have largely resolved. Women considering surgery should be informed of these effects.

Bridging the age gap in breast cancer: cluster randomized trial of two decision support interventions for older women with operable breast cancer on quality of life, survival, decision quality, and treatment choices.

BACKGROUND: Rates of surgery and adjuvant therapy for breast cancer vary widely between breast units. This may contribute to differences in survival. This cluster RCT evaluated the impact of decision support interventions (DESIs) for older women with breast cancer, to ascertain whether DESIs influenced quality of life, survival, decision quality, and treatment choice. METHODS: A multicentre cluster RCT compared the use of two DESIs against usual care in treatment decision-making in older women (aged at least ≥70 years) with breast cancer. Each DESI comprised an online algorithm, booklet, and brief decision aid to inform choices between surgery plus adjuvant endocrine therapy versus primary endocrine therapy, and adjuvant chemotherapy versus no chemotherapy. The primary outcome was quality of life. Secondary outcomes included decision quality measures, survival, and treatment choice. RESULTS: A total of 46 breast units were randomized (21 intervention, 25 usual care), recruiting 1339 women (670 intervention, 669 usual care). There was no significant difference in global quality of life at 6 months after the baseline assessment on intention-to-treat analysis (difference -0.20, 95 per cent confidence interval (C.I.) -2.69 to 2.29; P = 0.900). In women offered a choice of primary endocrine therapy versus surgery plus endocrine therapy, knowledge about treatments was greater in the intervention arm (94 versus 74 per cent; P = 0.003). Treatment choice was altered, with a primary endocrine therapy rate among women with oestrogen receptor-positive disease of 21.0 per cent in the intervention versus 15.4 per cent in usual-care sites (difference 5.5 (95 per cent C.I. 1.1 to 10.0) per cent; P = 0.029). The chemotherapy rate was 10.3 per cent at intervention versus 14.8 per cent at usual-care sites (difference -4.5 (C.I. -8.0 to 0) per cent; P = 0.013). Survival was similar in both arms. CONCLUSION: The use of DESIs in older women increases knowledge of breast cancer treatment options, facilitates shared decision-making, and alters treatment selection. Trial registration numbers: EudraCT 2015-004220-61 (https://eudract.ema.europa.eu/), ISRCTN46099296 (http://www.controlled-trials.com).

A phase I study of daily afatinib, an irreversible ErbB family blocker, in combination with weekly paclitaxel in patients with advanced solid tumours.

BACKGROUND: This phase I study evaluated afatinib, an irreversible ErbB family blocker, plus paclitaxel in patients with advanced solid tumours likely to express human epidermal growth factor receptor (HER1/EGFR) or HER2. METHODS: Oral afatinib was combined with intravenous paclitaxel (80mg/m(2); days 1, 8 and 15 every four weeks) starting at 20mg once daily and escalated to 40 and 50mg in successive cohorts of ⩾3 patients. The primary objective was to determine the maximum tolerated dose (MTD) of afatinib combined with paclitaxel. Secondary objectives included safety, pharmacokinetics and antitumour activity. RESULTS: Sixteen patients were treated. Dose-limiting toxicities with afatinib 50mg were fatigue and mucositis. The MTD was determined as afatinib 40mg with paclitaxel 80mg/m(2), which proved tolerable with repeated dosing. Frequent adverse events (AEs) included diarrhoea (94%), fatigue (81%), rash/acne (81%), decreased appetite (69%) and inflammation of mucosal membranes (69%); no grade 4 treatment-related AEs were observed. Five (31%) confirmed partial responses were observed in patients with non-small cell lung cancer (n=3), oesophageal cancer and cholangiocarcinoma; eight (50%) patients remained on study for ⩾6months. Pharmacokinetic parameters of afatinib and paclitaxel were similar for single administration or in combination. CONCLUSIONS: The MTD and recommended phase II dose of once-daily afatinib combined with paclitaxel 80mg/m(2) (days 1, 8 and 15 every four weeks) was 40mg. AEs at or below this dose were generally manageable with repeated dosing. No pharmacokinetic interactions were observed. This combination demonstrated promising antitumour activity. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00809133.

Phase III, randomized, placebo-controlled study of once-daily oral zibotentan (ZD4054) in patients with non-metastatic castration-resistant prostate cancer.

BACKGROUND: Standard treatment options are limited for the management of non-metastatic castration-resistant prostate cancer (CRPC). This study, part of the ENTHUSE (EndoTHelin A USE) phase III programme, evaluated the efficacy and safety of the oral specific endothelin (ET)A receptor antagonist zibotentan vs placebo in patients with non-metastatic CRPC (non-mCRPC). METHODS: This was a multicentre, randomized, double-blind, phase III study. Patients (n=1421) with non-mCRPC and biochemical progression (determined by rising serum PSA levels) were randomized to receive zibotentan 10 mg or placebo once daily. Based on the lack of efficacy signal in another ENTHUSE phase III study, an interim analysis was performed to determine whether the study was likely to achieve the co-primary objectives of improved overall survival (OS) and progression-free survival (PFS). RESULTS: Criteria for continuation of this study were not met. A total of 79 deaths and 293 progression events were recorded at final data cutoff. Zibotentan-treated patients did not significantly differ from placebo-treated patients for OS (hazard ratio (HR): 1.13; 95% confidence interval (CI): 0.73-1.76, P=0.589) or PFS (HR: 0.89; 95% CI: 0.71-1.12, P=0.330). The most commonly reported adverse events in zibotentan-treated patients were peripheral oedema (37.7%), headache (26.2%) and nasal congestion (24.9%); each occurred with >15% higher incidence than in the placebo group. CONCLUSIONS: This trial was terminated early because of failure at interim analysis of the efficacy data to meet the defined criteria for continuation. Owing to the absence of demonstrable survival benefits in the ENTHUSE clinical studies, zibotentan is no longer under investigation as a potential treatment for prostate cancer.

A Phase II, randomized, double-blind study of zibotentan (ZD4054) in combination with carboplatin/paclitaxel versus placebo in combination with carboplatin/paclitaxel in patients with advanced ovarian cancer sensitive to platinum-based chemotherapy (AGO-OVAR 2.14).

BACKGROUND: In platinum-sensitive relapsed ovarian cancer, paclitaxel plus carboplatin is a standard second-line treatment. Zibotentan (ZD4054) is an oral, specific ETA-receptor antagonist with demonstrated antitumour activity in xenograft models of human ovarian cancer. METHODS: In this Phase II, randomized, placebo-controlled study, patients with relapsed ovarian cancer sensitive to platinum-based chemotherapy received zibotentan 10mg or placebo once-daily, plus paclitaxel 175 mg/m(2) iv followed by carboplatin iv (AUC 5) on day 1 of every 3-week cycle for a maximum of eight cycles. The primary endpoint was progression-free survival (PFS), evaluated by Response Evaluation Criteria In Solid Tumours (RECIST). Secondary and exploratory endpoints included objective tumour response rate, tumour size, CA-125/RECIST progression, and safety and tolerability. RESULTS: A total of 120 patients were randomized (zibotentan: n=59; placebo: n=61). Addition of zibotentan 10mg/day to carboplatin and paclitaxel did not improve PFS compared with placebo (median PFS, 7.6 versus 10.0 months, respectively; HR=1.46, [80% CI: 1.10-1.94]; P=0.0870). No improvements in any of the secondary or exploratory efficacy endpoints were observed for patients receiving zibotentan compared with placebo. Median duration of total treatment exposure was 6.7 months. Total chemotherapy dose received was lower for zibotentan-treated versus placebo-treated patients (carboplatin: -16%; paclitaxel: -14%). The most common adverse events in the zibotentan arm were anaemia, nausea, alopecia, headache and neutropenia (43-48% of patients). CONCLUSIONS: Zibotentan 10mg/day plus carboplatin and paclitaxel did not result in an improvement in PFS compared with chemotherapy alone in patients with advanced ovarian cancer sensitive to platinum-based chemotherapy. No unexpected safety concerns were identified.

Synthesis of a new series of 4-aryl-1,4-dihydropyridines with calcium channel blocking and vasodilatory activity.

Synthesis of a new series of 4-aryl-1,4-dihydropyridines possessing potential calcium channel blocking activity along with good vasodilatory profile is reported. The compounds were synthesized using modified Hantzsch condensation of various aldehydes with methyl 3-aminocrotonate in the presence of a catalytic amount of trifluoroacetic acid and subsequent alkylation with various hydrochlorides of dialkylaminoalkyl chlorides. In vitro calcium channel blocking activity has been evaluated in cultures of neonatal rat cortical neurons by measuring the inhibitory response at L-type calcium channels activated by veratridine. Many compounds exhibited moderate to significant calcium channel blockade around 1 microM. The vasodilatory activity was assessed on isolated rat thoracic aortic rings precontracted by phenylephrine/KCl (30 mM). Most of the compounds produced a concentration-dependent inhibition of the contractile response.

Structure-activity relationships in a peptidic alpha7 nicotinic acetylcholine receptor antagonist.

alpha-Conotoxins are small disulfide-constrained peptide toxins which act as antagonists at specific subtypes of nicotinic acetylcholine receptors (nACh receptors). In this study, we analyzed the structures and activities of three mutants of alpha-conotoxin ImI, a 12 amino acid peptide active at alpha7 nACh receptors, in order to gain insight into the primary and tertiary structural requirements of neuronal alpha-conotoxin specificity. NMR solution structures were determined for mutants R11E, R7L, and D5N, resulting in representative ensembles of 20 conformers with average pairwise RMSD values of 0.46, 0.52, and 0.62 A from their mean structures, respectively, for the backbone atoms N, C(alpha), and C' of residues 2-11. The R11E mutant was found to have activity near that of wild-type ImI, while R7L and D5N demonstrated activities reduced by at least two orders of magnitude. Comparison of the structures reveals a common two-loop architecture, with variations observed in backbone and side-chain dihedral angles as well as surface electrostatic potentials upon mutation. Correlation of these structures and activities with those from previously published studies emphasizes that existing hypotheses regarding the molecular determinants of alpha-conotoxin specificity are not adequate for explaining peptide activity, and suggests that more subtle features, visualized here at the atomic level, are important for receptor binding. These data, in conjunction with reported characterizations of the acetylcholine binding site, support a model of toxin activity in which a single solvent-accessible toxin side-chain anchors the complex, with supporting weak interactions determining both the efficacy and the subtype specificity of the inhibitory activity.

Modulation of low-threshold T-type calcium channels by the five muscarinic receptor subtypes in NIH 3T3 cells.

The modulation of a transient T-type calcium current by the five muscarinic receptor subtypes, stably expressed in NIH 3T3 cells, was studied with the whole-cell patch-clamp technique. Voltage-step depolarizations applied to the NIH 3T3 cells revealed a low-voltage-activated (LVA) T-type calcium current that was inhibited by Ni2+ and unaffected by omega-conotoxin GVIA. In cells transfected with the m3 and m5 muscarinic receptors, application of acetylcholine (ACh) resulted in a pertussis-toxin-insensitive increase in peak T-type calcium current amplitude. The m3-induced atropine-sensitive increase in current amplitude was accompanied by a shift in the voltage dependence of activation to more hyperpolarized potentials. The increase in peak T-type calcium current amplitude and the shift in voltage dependence was mimicked by incubation with 500 microM 8-bromo-cAMP. Conversely, T-type calcium current amplitudes were reduced by incubation with 10 microM RpcAMPS, an inhibitor of cAMP-dependent protein kinase (PKA). Preincubation with 500 microM 8-bromo-cAMP or with 10 microM RpcAMPS abolished the increase in T-type calcium current amplitude previously noted on stimulation of the m3 muscarinic receptor by ACh. Application of ACh to NIH 3T3 cells stably transformed with the m1 muscarinic receptor resulted in no discernable change in T-type calcium current amplitude. However, on pre-incubation of the cells with calphostin C, an inhibitor of protein kinase C (PKC), application of ACh to the cells now resulted in a robust increase in T-type calcium current amplitude. Application of 500 nM PDBu, an activator of PKC, reduced the T-type calcium current amplitude. No significant changes in T-type calcium currents were observed on application of ACh to cells stably transfected with the m2 or m4 muscarinic receptors. However, after pre-incubation with forskolin, the m2 muscarinic receptor induced a decrease in T-type calcium current amplitude. Stimulation of the ml, m3 and m5 muscarinic receptors in the NIH 3T3 cell resulted in dose-dependent increases in the concentration of intracellular cAMP in comparison to control as determined by cAMP immunoassay. Conversely, stimulation of the m2 and m4 muscarinic receptors by carbachol resulted in a dose-dependent reduction in intracellular concentrations of cAMP, as compared with control basal levels. It is concluded that the m3 and m5 muscarinic receptors enhance T-type calcium channel activity. At least in the case of the m3 muscarinic receptor, the increased T-type channel activity appeared to be mediated via increased cAMP levels and subsequent activation of PKA. The lack of effect of the ml muscarinic receptor on the T-type calcium channel was probably due to the opposing actions of concomitant activation of both PKC and PKA. The physiological significance of these findings is discussed.

High-affinity kainate-type ion channels in rat cerebellar granule cells.

1. Patch-clamp recordings were made from rat cerebellar granule cells in primary culture. In cells pre-exposed to concanavalin A (ConA) to remove kainate receptor desensitization, concentration-response data for kainate showed two components. The EC50 value for the high-affinity component (4 microM) was consistent with activation of kainate-type channels. ConA enhanced the apparent potency of the kainate receptor ligand SYM 2081 by 100-fold. 2. In ConA-treated granule cells, currents evoked by 10 microM kainate were not significantly reduced by the AMPA receptor antagonist GYKI 53655, nor were these currents significantly reduced by the co-application of 100 microM AMPA. Currents activated by low concentrations of kainate in the presence of AMPA were completely inhibited by 10 microM La3+. 3. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicated that granule cells express both unedited (Q) and edited (R) versions of GluR5, with the majority of the GluR5 transcripts being unedited. In contrast, BluR6(R) was detected in seven cells and GluR6(Q) was detected in one granule cell. 4. Whole-cell current-voltage curves for kainate-type currents in granule cells were measured and the ratio of the slope conductances at +40 MV and -40 mV was used as an index of rectification. The mean +40 mV/-40 mV ratio determined from thirty-six granule cells was 1.3 +/- 0.1. Spectral density analysis of kainate-evoked whole-cell current noise gave values for the apparent single-channel conductance, gamma(noise), that were on average about 1 pS. 5. To compare further the properties of recombinant kainate channels with the native kainate-type channels in granule cells, we determined EC50 and gamma(noise) values for SYM 2081 in stable cell lines expressing either (GluR6(R) or GluR6(R) and KA2. Co-expression of KA2 with GluR6(R) shifts the EC50 and gamma(noise) values determined for SYM 2081 closer to the values typically found for native kainate-type channels in granule cells. 6. The results demonstrate that cerebellar granule cells in culture express functional kainate-type channels and that in most cells these channels show properties that are similar to those determined for heteromeric channels formed from GluR6(R) and KA2. However, the results also suggest that different granule cells express different repertoires of kainate-type channels with different, and perhaps variable, subunit composition.

Pneumonia: the impact of risk factors on the outcome of treatment with meropenem and ceftazidime.

This analysis comprises data pooled from two clinical trials of meropenem (0.5 g 8-hourly) versus ceftazidime (1 g 8-hourly) in hospitalized patients with community-acquired pneumonia. The clinical and bacteriological responses to treatment were assessed in relation to a range of risk factors previously linked to a poor clinical outcome. 393 patients (198 meropenem, 195 ceftazidime) were clinically evaluable while 230 (113 meropenem, 117 ceftazidime) were bacteriologically evaluable. Meropenem was highly effective, independent of associated risk factors, producing overall satisfactory clinical and bacteriological response rates at the end of therapy of 91.4% and 94.7%, respectively, similar to those produced by ceftazidime (90.3% and 92.3%, respectively). Clinical and bacteriological treatment outcome were similar in patients with up to three of the following key risk factors: age > or =65 years, male gender, serum urea >7 mmol/L, serum albumin <35 g/L and difficult-to-treat pathogens. Meropenem also achieved high clinical (85.7%) and bacteriological (89.3%) success rates in patients requiring ventilation, as did ceftazidime (81.6% and 87.1%, respectively). Both agents were highly effective against both Gram-negative and Gram-positive causative pathogens, including those organisms normally considered difficult to treat and typical of nosocomial pneumonia (e.g. Enterobacteriaceae, Staphylococcus aureus, Pseudomonas aeruginosa). Thus, meropenem and ceftazidime were highly effective in patients hospitalized with community-acquired pneumonia, irrespective of a number of concurrent risk factors (including those regarded as key risk factors). Furthermore, the analysis points to a role for meropenem 0.5 g 8-hourly in the treatment of nosocomial pneumonias that do not require intensive care unit admission and/or mechanical ventilation. Overall, this novel analysis of trial data suggests that incorporation of key risk factor endpoints into the initial design of pneumonia studies may prove to be a useful approach in defining appropriate antibiotic treatment for specific patient groups.

Inhibition of the L-type calcium channel by the five muscarinic receptors (m1-m5) expressed in NIH 3T3 cells.

Modulation of L-type calcium channels by the five cloned muscarinic receptors was studied by expression of the receptors in NIH 3T3 cells. Application of acetylcholine (ACh) to cells transfected with m1-m5 resulted in a reduction in the L-type calcium current amplitude. Elevations in intracellular cAMP concentrations induced by 8-bromo-cAMP or forskolin resulted in no discernible change in the L-type calcium current. In addition, treatment with Rp-adenosine 3',5'-cyclic monophosphothioate triethylamine (Rp-cAMPS), a protein kinase A (PKA) inhibitor, had no effect on the L-type currents. Conversely, application of phorbol dibutyrate, an activator of protein kinase C (PKC) or 8-bromo-cGMP, an activator of cGMP-dependent protein kinase (PKG), reduced the calcium currents. Incubation of the cells with KT5823, an inhibitor of PKG, resulted in a reduction of the response to 8-bromo-cGMP. The ACh-induced depression of L-type calcium current amplitude was sensitive to pertussis toxin (PTX) in cells transfected with the m2 or m4 receptor subtype. The m2-muscarinic-receptor-induced inhibition of the L-type calcium current was attenuated by preincubation of the cells with 8-bromo-cAMP and was unaffected by KT5823 or by calphostin C. The m1-muscarinic-receptor-induced inhibition of the L-type calcium conductance was insensitive to PTX treatment. However, the m1-induced response was blocked by preincubation of the cells with calphostin C. The present data indicate that the m2 (and possibly also the m4) muscarinic receptors inhibit the L-type calcium conductance by a reduction in cAMP concentration and that the m1 (and possibly also the m3 and m5) muscarinic receptors inhibit the L-type calcium channel via activation of PKC.

Enhancement of an L-type calcium current in AtT-20 cells; a novel effect of the m4 muscarinic receptor.

Activation of muscarinic receptors has been shown to inhibit L-type calcium conductances by mechanisms sensitive to pertussis toxin (PTX). In this study we show that agonist stimulation of the m4 muscarinic receptor leads to an increase in an L-type calcium conductance in the AtT-20 pituitary cell line, by a PTX-sensitive mechanism. The amplitude of the dihydropyridine (DHP)-sensitive or L-type calcium current was increased by acetylcholine (ACh), with no shift in the voltage dependence. This action of ACh was completely inhibited by PTX pre-treatment. Forskolin, cAMP and phorbol 12,13-dibutyrate reduced, while RpcAMPs, an inhibitor of cAMP-dependent protein kinase (PKA), increased the L-type calcium conductance. We propose that the m4 muscarinic receptor activates the L-type calcium channel by inhibition of adenylyl cyclase resulting in reduced cAMP levels and, hence, reduced PKA activity. This novel increase in calcium current via the m4 muscarinic receptor appears to reflect the coupling with an L-type channel of the D class, due to the sensitivity of the L-type calcium conductance to both DHPs and omega-conotoxin, and, thus, is distinct from the skeletal muscle and cardiac L-type channels of the C class previously studied.

Identification of myocardial proteins from two-dimensional gels by peptide mass fingerprinting.

Two-dimensional gels offer a powerful method for separating complex protein mixtures, but subsequent methods for analysing individual components, such as protein sequencing and Western immunoblotting, are laborious and slow. The identification of proteins can be accelerated by using a combination of protease digestion and matrix assisted laser desorption-mass spectrometry (MALDI-MS). The peptide mass spectrum of a protein represents a unique fingerprint determined by the amino acid sequence and the cleavage properties of the protease. Software has been developed so that peptide masses can be used to search a mass-based peptide database generated from established protein sequence databases. A list of the closest matching proteins is produced to allow identification of the sample. The strategy was applied to 52 protein spots from human myocardial tissue separated by two-dimensional electrophoresis (2-DE) gels and analysed blind. Conditions for optimal trypsin digestion of proteins electroblotted onto polyvinylidene difluoride (PVDF) membranes are described. Mass data were generated from both Coomassie Brilliant Blue and sulforhodamine B-stained proteins, though the former required destaining prior to digestion. Alkylation of cysteine and oxidation of methionine were significant modifications that influenced the successful identification of a protein spot. Examples are presented to illustrate the advantages and disadvantages of this approach.

Effect of veratridine on miniature endplate current amplitudes at the rat neuromuscular junction and acetylcholine uptake by Torpedo synaptic vesicles.

Veratridine produces a marked elevation in spontaneous quantal release from nerve endings through its ability to enhance sodium-channel activity, leading to sustained membrane depolarization. In the course of an electrophysiological investigation into the effects of vesamicol, an inhibitor of the synaptic vesicle acetylcholine transporter, on veratridine-induced acetylcholine release from rat motor nerve terminals we observed that veratridine itself has an effect on miniature endplate current amplitude distributions suggestive of an effect of the compound on the filling of cholinergic synaptic vesicles with acetylcholine. This effect of veratridine is release-dependent, being inhibited by either removal of extracellular calcium ions or by the addition of the sodium channel blocking toxin, tetrodotoxin. Biochemical studies using synaptic vesicles isolated from Torpedo electroplaque confirmed the ability of veratridine to directly inhibit the vesicular transport of acetylcholine. This appears to be a consequence of its ability to dissipate the trans-vesicular membrane proton gradient, which normally drives the active transport of acetylcholine into synaptic vesicles. We discuss how such an action of veratridine could lead to the observed release-dependent effects of the compound on electrophysiologically monitored spontaneous quantal acetylcholine release. The action of veratridine on cholinergic synaptic vesicles could be of considerable import when using this agent to elicit neurotransmitter release from either peripheral or central nerve endings.

Efficient gene transfer into human umbilical vein endothelial cells allows functional analysis of the human tissue factor gene promoter.

Tissue factor (TF) is a cellular receptor and cofactor for factor VII/VIIa which initiates the blood coagulation cascade. We have investigated the role of 5'-flanking DNA sequences in regulating the expression of the human TF gene in human umbilical vein endothelial cells (HUVEC). Using a chloramphenicol acetyltransferase (CAT) reporter gene, we attempted to transfect primary cultured HUVEC (passage 3-4) with calcium phosphate coprecipitation, DEAE Dextran, lipopolyamine-coated DNA or electroporation. Electroporation in HEPES-buffered saline of 1 x 10(7) cells at 200V and 250 microF was found to be optimal. Using these conditions, varying lengths of TF 5'-flanking sequences coupled to the CAT reporter gene were tested in transient expression studies. CAT expression corrected for variation in transfection efficiency and cell viability revealed that the sequences between -111 and +14 base pairs are essential for minimal transcriptional activity. This region contains consensus sequences for a TATA box and three Sp1 binding sites. A domain from -382 to -111bp, which contains two AP-1 consensus elements, promoted high levels of gene expression. This transcriptional activity was repressed by 50% with constructs containing sequences between -550 and -382 bp. A further 2-fold drop in transcription activity was attributed to the region between -948 and -550 bp. These results suggest that the basal transcription of the human TF gene in HUVEC is mediated through at least two negative regulatory elements upstream of the proximal promoter domain. The proximal promoter region which contains two AP-1 sites is essential for efficient transcription.

The influence of tamoxifen in vivo on the main natural anticoagulants and fibrinolysis.

The long-term effects of tamoxifen on alterations in haemostasis which could lead towards thrombosis were investigated in 149 women who were disease-free for at least 5 years following mastectomy for breast cancer. All participants were randomized to receive tamoxifen as a post-surgical adjuvant treatment (89 patients, treated group) or not (60 patients, controls) for at least 2 years. 5.62% of the cases treated with tamoxifen suffered a venous thrombosis, while no thromboembolism was reported in the control group. No significant differences were observed between groups in the global clotting times, fibrinogen, fibrinolytic factors, or in the concentration of the main natural anticoagulants, antithrombin III (AT-III), protein C(PC) and protein S (PS). However, when the treated group was sub-divided, current users (n = 18) of the drug (median treatment duration 72 months) had significantly lower AT-III (P < 0.05) and PC (P < 0.05) activities, together with higher levels of plasminogen activity (P < 0.01) and tissue plasminogen activator antigen (P < 0.01), compared with 71 ex-users (who mostly received treatment for 2 years) and controls. We conclude that long-term treatment with tamoxifen for 2 or more years tends to reduce both AT-III and PC, a situation possibly predisposing towards thrombosis. Monitoring haemostasis in tamoxifen-treated breast cancer patients is therefore advisable.

Congenital thrombophilia among patients with venous thromboembolism.

During a 3-year period we studied 393 adult patients (382 of whom were unrelated) with a history of acute venous thromboembolism. A congenital deficiency state known to predispose to thrombosis was found in 27.2%. Of these, most were due to deficiencies of protein C (9.2%), protein S (7.6%), antithrombin III (5%) or to increased plasma PAI-1 concentration (3.1%) which, in the absence of any known factor that predisposes towards thrombosis, results in a diminished fibrinolytic activity. There was a characteristic pattern between the age of onset (mean 34 years) of thrombosis and individual protein deficiency. Thrombosis appeared spontaneously in 73% of cases with recurrence in 80%. In contrast, in the remaining unrelated patients, 138 (35.1%) in whom venous thromboembolism was secondary and occurred at a mean age of 43 years, and in the other 140 (35.6%) who suffered thromboembolism spontaneously at a later age (mean age 55), there was no permanent protein deficiency state or alteration in fibrinolytic activity and thrombosis recurrence was lower (53.6% and 20.7% respectively). Of the 393 patients, deep vein thrombosis was the most common manifestation; however, in congenital thrombophilia, thrombosis of visceral vessels and Raynaud's syndrome (6%) were also detected.

Use of low molecular weight heparin in pregnancy.

In a controlled study of 15 pregnant patients undergoing therapeutic termination of pregnancy, seven received subcutaneously 5,000 anti-FXa units of low molecular weight (LMW) heparin 15 and 3 h prior to the termination, and eight patients acted as controls. Paired maternal and fetal blood samples were taken (before or immediately after the termination) for assay of heparin activity by a chromogenic anti-FXa method sensitive to levels of 0.02 anti-FXa U/ml. LMW heparin was detected in all maternal samples of the test patients but was not detected in any of the fetal samples. The use of LMW heparin as a thromboprophylactic agent was then evaluated in 11 patients who were known to have a severe thromboembolic tendency, had suffered recurrent miscarriages and had responded poorly to conventional anticoagulation (oral anticoagulant, conventional heparin). All patients receiving LMW heparin in thromboprophylactic doses completed uneventful pregnancies and gave birth to healthy babies (three for the first time) without complication. Bone density scans performed in all patients shortly after the delivery showed normal mineral mass. We conclude that LMW heparin does not cross the placental barrier, and in addition offers satisfactory antithrombotic protection for both maternal and placental circulation. In addition, this study provides preliminary data from 11 patients suggesting LMWH may not give rise to maternal osteoporosis, a finding that now needs further investigation.

The effects of vesamicol on trains of endplate currents and on focally recorded nerve terminal currents at mammalian neuromuscular junctions.

1. The effects of vesamicol, an inhibitor of vesicular acetylcholine (ACh) storage, were studied on trains of endplate currents (e.p.cs) in the cut rat hemidiaphragm nerve-muscle preparation and on trains of focally recorded nerve terminal current waveforms in the mouse triangularis sterni nerve-muscle preparation. 2. In the rat, 0.1 and 1 microM (-)-vesamicol produced an enhancement of the rundown of e.p.c. amplitudes during trains of high frequency (50 Hz) nerve stimulation. However, 1 microM (+)-vesamicol had no effect on the rundown of e.p.c. amplitudes. 3. In the mouse, high concentrations of (-)-vesamicol (10-100 microM) produced a concentration- and stimulation-dependent decrease in the amplitude of the second negative-going deflection of focally recorded nerve terminal current waveforms. 4. At 1 mM, (-)-vesamicol produced a stimulation-independent decrease in the amplitude of the first negative-going deflection of the nerve terminal current waveforms, an increase in signal delay and evidence of nerve conduction failure. These all indicate a local anaesthetic-like block of nodal Na(+)-channels. 5. In contrast to its effects on trains of e.p.cs, the effects of vesamicol on the nerve terminal current waveforms were not stereoselective, the (+)-isomer being equipotent with the (-)-isomer. 6. Low concentrations of the Na(+)-channel blocking toxin, tetrodotoxin (15-60 nM), produced similar changes in the focally recorded nerve terminal current waveforms to those seen with vesamicol. 7. It is concluded that the stereoselective rundown of e.p.c. amplitudes produced by (-)-vesamicol is due to an effect, either direct or indirect, on ACh mobilization within motor nerve terminals. Furthermore, in mammalian species, the inhibitory effects of vesamicol on nodal Na+-channels which are seen at high concentrations do not contribute to the principal neuromuscular effects of the compound.

The "staircase test": a measure of independent forelimb reaching and grasping abilities in rats.

A novel reaching test for the rat has been developed to assess the independent use of forelimbs in skilled reaching and grasping tasks. The apparatus is a plexiglas box with a removable baited double staircase. Food pellets are placed on the staircase and presented bilaterally at 7 graded stages of reaching difficulty to provide objective measures of side bias, maximum forelimb extension and grasping skill. In the present experiment, the apparatus was used to assess the reaching performance of rats following unilateral lesions of the sensorimotor cortex, unilateral lesions of the posterior cortex or bilateral lesions of the olfactory bulbs. The task has the advantage of objective over rating measurement, and the simplicity of the apparatus permits many animals to be tested concurrently.