Rapamycin, an inhibitor of mammalian target of rapamycin complex-1 (mTORC1), reduces platelet spreading, thrombus stability, and clot retraction. Despite an important role of mTORC1 in platelet function, little is known about how it is regulated. The objective of this study was to determine the signaling pathways that regulate mTORC1 in human platelets.
Mammalian target of rapamycin complex-1 activation was assessed by measuring the phosphorylation of its downstream substrate ribosomal S6 kinase 1 (p70S6K).
Thrombin or the protein kinase C (PKC) activator phorbal 12-myristate 13-acetate stimulated activation of mTORC1 in a PKC-dependent, Akt-independent manner that correlated with phosphorylation of tuberin/tuberous sclerosis 2 (TSC2) (Ser939 and Thr1462). In contrast, insulin-like growth factor 1 (IGF-1)–stimulated TSC2 phosphorylation was completely dependent on phosphoinositide 3 kinase (PI3 kinase)/Akt but did not result in any detectable mTORC1 activation. Early (Ser939 and Thr1462) and late (Thr1462) TSC2 phosphorylation in response to thrombin were directly PKC dependent, whereas later TSC2 (Ser939) and p70S6K phosphorylation were largely dependent on paracrine signaling through P2Y12. PKC-mediated adenosine diphosphate (ADP) secretion was essential for thrombin-stimulated mTORC1 activation, as (i) ADP rescued p70S6K phosphorylation in the presence of a PKC inhibitor and (ii) P2Y12 antagonism prevented thrombin-mediated mTORC1 activation. Rescue of mTORC1 activation with exogenous ADP was completely dependent on the Src family kinases but independent of PI3 kinase/Akt. Interestingly, although inhibition of Src blocked the ADP rescue, it had little effect on thrombin-stimulated p70S6K phosphorylation under conditions where PKC was not inhibited.
These results demonstrate that thrombin activates the mTORC1 pathway in human platelets through PKC-mediated ADP secretion and subsequent activation of P2Y12, in a manner largely independent of the canonical PI3 kinase/Akt pathway.
P2Y12 purinoceptor; platelets; protease-activated receptors; protein kinase C; target of rapamycin complex 1
The β-tropomyosin gene encodes a component of the sarcomeric thin filament. Rod-shaped dimers of tropomyosin regulate actin-myosin interactions and β-tropomyosin mutations have been associated with nemaline myopathy, cap myopathy, Escobar syndrome and distal arthrogryposis types 1A and 2B. In this study, we expand the allelic spectrum of β-tropomyosin-related myopathies through the identification of a novel β-tropomyosin mutation in two clinical contexts not previously associated with β-tropomyosin. The first clinical phenotype is core-rod myopathy, with a β-tropomyosin mutation uncovered by whole exome sequencing in a family with autosomal dominant distal myopathy and muscle biopsy features of both minicores and nemaline rods. The second phenotype, observed in four unrelated families, is autosomal dominant trismus-pseudocamptodactyly syndrome (distal arthrogryposis type 7; previously associated exclusively with myosin heavy chain 8 mutations). In all four families, the mutation identified was a novel 3-bp in-frame deletion (c.20_22del) that results in deletion of a conserved lysine at the seventh amino acid position (p.K7del). This is the first mutation identified in the extreme N-terminus of β-tropomyosin. To understand the potential pathogenic mechanism(s) underlying this mutation, we performed both computational analysis and in vivo modelling. Our theoretical model predicts that the mutation disrupts the N-terminus of the α-helices of dimeric β-tropomyosin, a change predicted to alter protein–protein binding between β-tropomyosin and other molecules and to disturb head-to-tail polymerization of β-tropomyosin dimers. To create an in vivo model, we expressed wild-type or p.K7del β-tropomyosin in the developing zebrafish. p.K7del β-tropomyosin fails to localize properly within the thin filament compartment and its expression alters sarcomere length, suggesting that the mutation interferes with head-to-tail β-tropomyosin polymerization and with overall sarcomeric structure. We describe a novel β-tropomyosin mutation, two clinical-histopathological phenotypes not previously associated with β-tropomyosin and pathogenic data from the first animal model of β-tropomyosin-related myopathies.
nemaline; myopathies; muscle and nerve pathology; mutation; neuromuscular disorders
The purpose of this study was to determine the immediate and long-term effects of intraplaque hemorrhage (IPH) on plaque progression in the carotid artery.
Previous studies have associated IPH in the carotid artery with more rapid plaque progression. However, the time course and long-term effect remain unknown. Carotid magnetic resonance imaging (MRI) is a non-invasive imaging technique that has been validated with histology for the accurate in vivo detection of IPH and measurement of plaque burden.
Asymptomatic subjects with 50–79% carotid stenosis underwent carotid MRI at baseline and then serially every 18 months for a total of 54 months. Subjects with IPH present in at least one carotid artery at 54 months were selected. Subsequently, presence/absence of IPH and wall volume were determined independently in all time points for both sides. A piecewise progression curve was fit using linear mixed model to compare progression rates defined as annualized changes in wall volume between periods defined by their relationship to IPH development.
From 14 patients that showed IPH at 54 months, 12 arteries were found to have developed IPH during the study period. The progression rates were −20.5±13.1, 20.5±13.6 and 16.5±10.8 mm3/year before, during and after IPH development, respectively. The progression rate during IPH development tended to be higher than the period before (p=0.080), but comparable to the period after (p=0.845). The progression rate in the combined period during/after IPH development was 18.3±6.5 mm3/year, which indicated significant progression (p=0.008 compared to a slope of 0) and was higher than the period before IPH development (p=0.018). No coincident ischemic events were noted for new IPH.
The development of IPH posed an immediate and long-term promoting effect on plaque progression. IPH appears to alter the biology and natural history of carotid atherosclerosis. Early identification of patients with IPH may prove invaluable in optimizing management to minimize future sequelae.
carotid arteries; intraplaque hemorrhage; atherosclerosis; magnetic resonance imaging
For late gadolinium enhancement (LGE) cardiovascular magnetic resonance (CMR) assessment of atrial scar to guide management and targeting of ablation in atrial fibrillation (AF), an objective, reproducible method of identifying atrial scar is required.
To describe an automated method for operator-independent quantification of LGE that correlates with colocated endocardial voltage and clinical outcomes.
LGE CMR imaging was performed at 2 centers, before and 3 months after pulmonary vein isolation for paroxysmal AF (n = 50). A left atrial (LA) surface scar map was constructed by using automated software, expressing intensity as multiples of standard deviation (SD) above blood pool mean. Twenty-one patients underwent endocardial voltage mapping at the time of pulmonary vein isolation (11 were redo procedures). Scar maps and voltage maps were spatially registered to the same magnetic resonance angiography (MRA) segmentation.
The LGE levels of 3, 4, and 5SDs above blood pool mean were associated with progressively lower bipolar voltages compared to the preceding enhancement level (0.85 ± 0.33, 0.50 ± 0.22, and 0.38 ± 0.28 mV; P = .002, P < .001, and P = .048, respectively). The proportion of atrial surface area classified as scar (ie, >3 SD above blood pool mean) on preablation scans was greater in patients with postablation AF recurrence than those without recurrence (6.6% ± 6.7% vs 3.5% ± 3.0%, P = .032). The LA volume >102 mL was associated with a significantly greater proportion of LA scar (6.4% ± 5.9% vs 3.4% ± 2.2%; P = .007).
LA scar quantified automatically by a simple objective method correlates with colocated endocardial voltage. Greater preablation scar is associated with LA dilatation and AF recurrence.
2D, 2-dimensional; AF, atrial fibrillation; CMR, cardiovascular magnetic resonance; ECG, electrocardiogram; LA, left atrial/atrium; LGE, late gadolinium enhancement; MRA, Magnetic resonance angiography; PAF, paroxysmal atrial fibrillation; RF, radiofrequency; SD, standard deviation; Atrial fibrillation; Delayed-enhancement magnetic resonance imaging; Radiofrequency ablation
Alginate calcification has been previously reported clinically and during animal implantation; however no study has investigated the mechanism, extensively characterized the mineral, or evaluated multiple methods to regulate or eliminate mineralization. In the present study, alginate calcification was first studied in vitro: calcium-crosslinked alginate beads sequestered surrounding phosphate while forming traces of hydroxyapatite. Calcification in vivo was then examined in nude mice using alginate microbeads with and without adipose stem cells (ASCs). Variables included the delivery method, site of delivery, sex of the animal, time in vivo, crosslinking solution, and method of storage prior to delivery. Calcium-crosslinked alginate microbeads mineralized when injected subcutaneously or implanted intramuscularly after 1–6 months. More extensive analysis with histology, microCT, FTIR, XRD, and EDS showed calcium phosphate deposits throughout the microbeads with surface mineralization that closely matched hydroxyapatite found in bone. Incorporating 25 mM bisphosphonate reduced alginate calcification whereas using barium chloride eliminated mineralization. Buffering the crosslinking solution with HEPES at pH 7.3 while washing and storing samples in basal media prior to implantation also eliminated calcification in vivo. This study shows that alginate processing prior to implantation can significantly influence bulk hydroxyapatite formation and presents a method to regulate alginate calcification.
Calcification; Alginate; Microencapsulation; Adipose stem cell microbeads; Bone tissue engineering
Cross-relaxation imaging (CRI) is a quantitative magnetic resonance technique that measures the kinetic parameters of magnetization transfer between protons bound to water and protons bound to macromolecules. In this study, in vivo, four-parameter CRI of normal rat brains (N=5) at 3.0 T was first directly compared to histology. The bound pool fraction, f, was strongly associated with myelin density (Pearson’s r = 0.99, p <0.001). The correlation persisted in separate analyses of gray matter (GM; r = 0.89, p =0.046) and white matter (WM; r = 0.97, p =0.029). Subsequently, a new time-efficient approach for solely capturing the whole-brain parametric map of f was proposed, validated with histology, and used to estimate myelin density. Since the described approach for the rapid acquisition of f applied constraints to other CRI parameters, a theoretical analysis of error was performed. Estimates of f in normal and pathologic tissue were expected to have <10% error. A comparison of values for f obtained from the traditional four-parameter fit of CRI data versus the proposed rapid acquisition of f was within this expected margin for in vivo rat brain gliomas (N=4; mean ± SE; 3.9 ± 0.2% vs. 4.0 ± 0.2%, respectively). In both whole-brain f maps and myelin density maps, replacement of normal GM and WM by proliferating and invading tumor cells could be readily identified. The rapid, whole-brain acquisition of the bound pool fraction may provide a reliable method for detection of glioma invasion in both GM and WM during animal and human imaging.
bound pool fraction; cross-relaxation imaging; glioma; magnetization transfer ratio; myelin; quantitative magnetization transfer; rat brain
Non-steroidal anti-inflammatory drugs (NSAIDs) contribute to gastrointestinal ulcer formation by inhibiting epithelial cell migration and mucosal restitution; however, the drug-affected signaling pathways are poorly defined. We investigated whether NSAID inhibition of intestinal epithelial migration is associated with depletion of intracellular polyamines, depolarization of membrane potential (Em) and altered surface expression of K+ channels. Epithelial cell migration in response to the wounding of confluent IEC-6 and IEC-Cdx2 monolayers was reduced by indomethacin (100 μM), phenylbutazone (100 μM) and NS-398 (100 μM) but not by SC-560 (1 μM). NSAID-inhibition of intestinal cell migration was not associated with depletion of intracellular polyamines. Treatment of IEC-6 and IEC-Cdx2 cells with indomethacin, phenylbutazone and NS-398 induced significant depolarization of Em, whereas treatment with SC-560 had no effect on Em. The Em of IEC-Cdx2 cells was: −38.5±1.8 mV under control conditions; −35.9±1.6 mV after treatment with SC-560; −18.8±1.2 mV after treatment with indomethacin; and −23.7±1.4 mV after treatment with NS-398. Whereas SC-560 had no significant effects on the total cellular expression of Kv1.4 channel protein, indomethacin and NS-398 decreased not only the total cellular expression of Kv1.4, but also the cell surface expression of both Kv1.4 and Kv1.6 channel subunits in IEC-Cdx2. Both Kv1.4 and Kv1.6 channel proteins were immunoprecipitated by Kv1.4 antibody from IEC-Cdx2 lysates, indicating that these subunits co-assemble to form heteromeric Kv channels. These results suggest that NSAID inhibition of epithelial cell migration is independent of polyamine-depletion, and is associated with depolarization of Em and decreased surface expression of heteromeric Kv1 channels.
To determine if better flow-suppression can meaningfully improve the reproducibility of measurements associated with carotid atherosclerotic disease, particularly for lumen and wall areas.
Eighteen subjects with carotid artery stenosis identified by duplex ultrasound (11 with 16–49% stenosis; 7 with 50–79% stenosis) underwent two carotid MRI examinations on a 3T scanner with a 4-channel phased array coil. High-resolution intermediate-weighted TSE (TR/TE=4000/8.5ms, 0.55mm in-plane resolution, 2mm slice thickness, 16 slices, 3min scan time) with two different flow suppression techniques (mDIR and MSDE) were obtained separately. For each subject, bilateral arteries were reviewed. One radiologist blinded to time points, flow suppression techniques, and clinical information, measured the arterial lumen area, wall area and total vessel wall area.
Compared to mDIR, the MSDE technique had a smaller inter-scan standard deviation (SD) in lumen (SD: 3.6 vs. 5.2 mm2, p=0.02), wall area measurements (SD: 4.5 vs. 6.4 mm2, p=0.02) and total vessel area measurement (SD: 4.4 vs. 4.9 mm2, p=0.07), and a trend towards smaller SD in total vessel area measurement (SD: 4.4 vs. 4.9 mm2, p=0.07).
The results from this study demonstrated that vessel wall imaging could quantify atherosclerotic plaque measurements more reliably with improved blood suppression technique. This relationship between flow suppression efficiency and reproducibility of plaque measurements is important as more reliable area measurements will be useful in clinical diagnosis and in serial MRI studies that monitor carotid atherosclerotic lesion progression and regression.
Vessel wall MRI; Reproducibility; MSDE; mDIR
We sought to identify clinical and/or plaque characteristics that affect atherosclerotic disease progression and arterial remodeling in the carotid artery with subclinical stenosis.
Increasing severity of stenosis has been associated with a higher risk of stroke. Factors that drive subclinical lesions to become stenotic plaques remain ambiguous. Carotid magnetic resonance imaging (MRI) has been validated with histology to accurately quantify in vivo arterial morphology and plaque composition.
A total of 67 asymptomatic participants with 16% to 49% carotid stenosis as demonstrated by duplex ultrasonography were imaged at 1.5-T with a carotid MRI protocol at baseline and at 18-month follow-up. Clinical and/or intra-arterial metrics with a significant association with change in plaque burden during multivariate analysis were evaluated for effects on lumen, wall, and total vessel volume.
From multiple regression analysis, intraplaque hemorrhage (IPH) (p < 0.001) and statin therapy (p = 0.015) were identified as key determinants of change in plaque burden. The group with IPH compared with the group without IPH demonstrated luminal narrowing, with a mean ± SD decrease in lumen volume (−24.9 ± 21.1 mm3/year vs. −0.5 ± 26.9 mm3/year; p = 0.005), a larger increase in wall volume (44.1 ± 36.1 mm3/year vs. 0.8 ± 34.5 mm3/year; p < 0.001), and no difference in total vessel volume (19.3 ± 27.4 mm3/year vs. 0.4 ± 42.4 mm3/year; p = 0.15). The nonstatin group compared with the statin group demonstrated outward remodeling, with an increase in wall volume (22.4 ± 35.6 mm3/year3/year vs. 0.9 ± 38.0 mm3/year; p = 0.026) and total vessel volume (19.2 ± 36.9 mm3/year vs. −4.9 ± 40.4 mm3/year; p = 0.019) and no difference in lumen volume (−5.8 ± 26.6 mm3/year vs. −3.2 ± 29.5 mm3/year; p = 0.72).
IPH may represent an indication of accelerated plaque growth and impending luminal compromise in the subclinical carotid artery. Statin therapy may stabilize lesions by slowing or halting lesion progression. This phase of plaque stenosis (16% to 49%) may be a critical stage for intrinsic and extrinsic factors to affect the atherosclerotic disease process.
atherosclerosis; carotid arteries; magnetic resonance imaging; remodeling
Histological studies suggest associations between hemorrhage and large lipid-rich/necrotic cores with thin or ruptured fibrous caps in advanced atherosclerosis. We investigated these associations in carotid arteries with mild to severe stenosis by in-vivo 3T MRI.
Methods and results
Seventy-seven patients with ≥50% carotid stenosis in at least one side by duplex ultrasound underwent bilateral multi-contrast carotid MRI scans. Measurements for wall and lipid-rich/necrotic core sizes, presence of hemorrhage and fibrous cap status (classified as intact thick, intact thin or ruptured) were recorded. Arteries with poor image quality, occlusion or no detectable lipid-rich/necrotic core were excluded. For the 798 MRI slices included, multivariate ordinal regression analysis demonstrated larger %lipid-rich/necrotic core (odds ratio for 10% increase, 1.49; p=0.02) and presence of hemorrhage (odds ratio, 5.91; p<0.001) were independently associated with a worse (intact thin or ruptured) stage of fibrous cap status. For artery-based multivariate analysis, a larger maximum %lipid-rich/necrotic core and presence of hemorrhage independently associated with worse fibrous cap status (p<0.001, for both). No hemorrhage was detected in arteries with thick fibrous caps.
Hemorrhage and larger %lipid-rich/necrotic core were independently associated with a thin or ruptured fibrous cap status at an early to advanced stage of carotid atherosclerosis.
magnetic resonance imaging; carotid artery; fibrous cap rupture; hemorrhage; lipid-rich necrotic core
To investigate the dependence of contrast-enhanced MRI of carotid artery atherosclerotic plaque on use of gadobenate dimeglumine versus gadodiamide.
Materials and Methods
15 subjects with carotid atherosclerotic plaque were imaged with 0.1 mmol/kg of each agent. For arteries with interpretable images, the areas of the lumen, wall, and necrotic core and overlying fibrous cap (when present) were measured, as were the percent enhancement and contrast-to-noise ratio (CNR). A kinetic model was applied to dynamic imaging results to determine the fractional plasma volume vp and contrast agent transfer constant Ktrans.
For 12 subjects with interpretable images, the agent used did not significantly impact any area measurements or the presence or absence of necrotic core (p>0.1 for all). However, the percent enhancement was greater for the fibrous cap (72% vs. 54%; p<0.05) necrotic core (51% vs. 42%; p=0.12), and lumen (42% vs. 63%; p<0.05) when using gadobenate dimeglumine, although no apparent difference in CNR was found. Additionally, Ktrans was lower when using gadobenate dimeglumine (0.0846 min−1 vs. 0.101 min−1; p<0.01), although vp showed no difference (9.5% vs. 10.1%; p=0.39).
Plaque morphology measurements are similar with either contrast agent, but quantitative enhancement characteristics, such as percent enhancement and Ktrans, differ.
contrast-enhanced MRI; atherosclerosis; gadobenate dimeglumine; gadodiamide; dynamic MRI
A&E departments are key points of contact for many people with mental health problems. Various models of care have been developed in A&E departments for delivering mental health services, but few have been assessed for effectiveness. The present study aimed to assess the impact of a dedicated A&E psychiatric nurse service on several outcomes relevant to patients and clinicians.
A crossover design was used to introduce a dedicated psychiatric nurse service (comprising four experienced community psychiatric nurses) into two busy UK A&E departments. Standardised assessments were completed for each patient, and a random sample of these independently assessed for quality. Data were also collected on the number of patients assessed, psychiatric nurse time employed, waiting times, onward referrals, repeat attendances, patient satisfaction, and staff views.
A&E staff referred about a third of patients judged to have mental health problems to the psychiatric nurse service; approximately half of those assessed had a psychiatric history. On average, assessments took 60 min and over 90% of the formulated management plans were judged appropriate by independent assessors. The psychiatric nurse intervention had little impact on waiting times or satisfaction levels for mental health patients, although there was evidence of a change in onward referral patterns.
Psychiatric nurse assessment services have been introduced in many A&E departments, although the evidence base for the effectiveness of this development is not well established. This study presents evidence that psychiatric nurses can provide an accurate assessment and referral service with advantages for patient care.
liaison psychiatric services; mental health; psychiatric assessment and treatment; psychiatric nursing
Carotid atherosclerotic ulceration is a significant source of stroke. This study evaluates the efficacy of adding longitudinal black-blood (BB) cardiovascular magnetic resonance (CMR) angiography to cross-sectional CMR images in the identification of carotid atherosclerotic ulceration.
Thirty-two subjects (30 males and two females with ages between 48 and 83 years) scheduled for carotid endarterectomy were imaged on a 1.5T GE Signa scanner using multisequence [3D time-of-flight, T1, proton density, T2, contrast enhanced T1], cross-sectional CMR images and longitudinal BB CMR angiography (0.625 × 0.625 mm/pixel). Two rounds of review (round 1: cross-sectional CMR images alone and round 2: cross-sectional CMR images plus longitudinal BB CMR angiography) were conducted for the presence and volume measurements of ulceration. Ulceration was defined as a distinct depression into the plaque containing blood flow signal on cross-sectional CMR and longitudinal BB CMR angiography.
Of the 32 plaques examined by histology, 17 contained 21 ulcers. Using the longitudinal BB CMR angiography sequence in addition to the cross-sectional CMR images in round 2, the sensitivity improved to 80% for ulcers of at least 6 mm3 in volume by histology and 52.4% for all ulcers, compared to 30% and 23.8% in round 1, respectively. There was a slight decline in specificity from 88.2% to 82.3%, though both the positive and negative predictive values increased modestly from 71.4% to 78.6% and from 48.4% to 58.3%, respectively.
The addition of longitudinal BB CMR angiography to multisequence cross-sectional CMR images increases accuracy in the identification of carotid atherosclerotic ulceration.
We sought to determine differences with cardiovascular magnetic resonance (CMR) in the morphology and composition of the carotid arteries between individuals with angiographically-defined obstructive coronary artery disease (CAD, ≥ 50% stenosis, cases) and those with angiographically normal coronaries (no lumen irregularities, controls).
Methods and results
191 participants (50.3% female; 50.8% CAD cases) were imaged with a multi-sequence, carotid CMR protocol at 1.5T. For each segment of the carotid, lumen area, wall area, total vessel area (lumen area + wall area), mean wall thickness and the presence or absence of calcification and lipid-rich necrotic core were recorded bilaterally. In male CAD cases compared to male controls, the distal bulb had a significantly smaller lumen area (60.0 ± 3.1 vs. 79.7 ± 3.2 mm2, p < 0.001) and total vessel area (99.6 ± 4.0 vs. 119.8 ± 4.1 mm2; p < 0.001), and larger mean wall thickness (1.25 ± 0.03 vs. 1.11 ± 0.03 mm; p = 0.002). Similarly, the internal carotid had a smaller lumen area (37.5 ± 1.8 vs. 44.6 ± 1.8 mm2; p = 0.006) and smaller total vessel area (64.0 ± 2.3 vs. 70.9 ± 2.4 mm2; p = 0.04). These metrics were not significantly different between female groups in the distal bulb and internal carotid or for either gender in the common carotid. Male CAD cases had an increased prevalence of lipid-rich necrotic core (49.0% vs. 19.6%; p = 0.003), while calcification was more prevalent in both male (46.9% vs. 17.4%; p = 0.002) and female (33.3% vs. 14.6%; p = 0.031) CAD cases compared to controls.
Males with obstructive CAD compared to male controls had carotid bulbs and internal carotid arteries with smaller total vessel and lumen areas, and an increased prevalence of lipid-rich necrotic core. Carotid calcification was related to CAD status in both males and females. Carotid CMR identifies distinct morphological and compositional differences in the carotid arteries between individuals with and without angiographically-defined obstructive CAD.
Evidence supports the use of exercise for chronic low back pain (CLBP); however, adherence is often poor due to ongoing pain. Auricular acupuncture is a form of pain relief involving the stimulation of points on the outer ear corresponding with specific body parts. It may be a useful adjunct to exercise in managing CLBP; however, there is only limited evidence to support its use with this patient group.
This study was designed to test the feasibility of an assessor-blind randomised controlled trial which assess the effects on clinical outcomes and exercise adherence of adding manual auricular acupuncture to a personalised and supervised exercise programme (PEP) for CLBP. No sample size calculation has been carried out as this study aims to identify CLBP referral rates within the catchment area of the study site. The researchers aim to recruit four cohorts of n = 20 participants to facilitate a power analysis for a future randomised controlled trial. A computer generated random allocation sequence will be prepared centrally and used to allocate participants by cohort to one of the following interventions: 1) six weeks of PEP plus manual auricular acupuncture; 2) six weeks of PEP alone. Both groups will also complete a further six weeks of self-paced exercise with telephone follow-up support. In addition to a baseline and exit questionnaire at the beginning and end of the study, the following outcomes will be collected at baseline, and after 7, 13 and 25 weeks: pain frequency and bothersomeness, back-specific function, objective assessment and recall of physical activity, use of analgesia, perceived self-efficacy, fear avoidance beliefs, and beliefs about the consequences of back pain. Since this is a feasibility study, significance tests will not be presented, and treatment effects will be represented by point estimates and confidence intervals. For each outcome variable, analysis of covariance will be performed on the data, conditioning on the baseline value.
The results of this study investigating the adjuvant effects of auricular acupuncture to exercise in managing CLBP will be used to inform the design of a future multi-centre randomised controlled trial.
Current Controlled Trials ISRCTN94142364.
The expression of hypoxia-regulated genes promotes an aggressive tumour phenotype and is associated with an adverse cancer treatment outcome. Thymidine phosphorylase (TP) levels increase under hypoxia, but the protein has not been studied in association with hypoxia in human tumours. An investigation was made, therefore, of the relationship of tumour TP with hypoxia, the expression of other hypoxia-associated markers and clinical outcome. This retrospective study was carried out in patients with locally advanced cervical carcinoma who underwent radiotherapy. Protein expression was evaluated with immunohistochemistry. Hypoxia was measured using microelectrodes and the level of pimonidazole binding. There was no relationship of TP expression with tumour pO2 (r=−0.091, P=0.59, n=87) or pimonidazole binding (r=0.13, P=0.45, n=38). There was no relationship between TP and HIF-1α, but there was a weak borderline significant relationship with HIF-2α expression. There were weak but significant correlations of TP with the expression of VEGF, CA IX and Glut-1. In 119 patients, the presence of TP expression predicted for disease-specific (P=0.032) and metastasis-free (P=0.050) survival. The results suggest that TP is not a surrogate marker of hypoxia, but is linked to the expression of hypoxia-associated genes and has weak prognostic power.
thymidine phosphorylase; hypoxia; hypoxia-inducible factor; cervix
The Bcl-2 family of apoptotic regulators is thought to play an essential role in cancer development and influence the sensitivity of tumour cells to radiotherapy. Bid is an abundantly expressed Bcl-2 family protein playing a central role in various pathways of apoptosis by integrating and converging signals at the mitochondria. The relevance of apoptotic modulation by Bcl-2 and related proteins in tumour development and radiation response for human tumours remains undefined. Therefore, a study was made regarding the expression of Bid in patients with locally advanced cervix carcinoma who received radiotherapy. Bid expression was assessed using immunohistochemistry in pretreatment archival biopsies from 98 patients. The data were correlated with clinicopathologic characteristics and treatment outcome. Pretreatment tumour radiosensitivity data were available for 60 patients. Strong Bid expression was associated with a patient age less than the median of 52 years (P=0.034) and poor metastasis-free survival. In multivariate analysis, after allowing for stage, Bid expression was a significant prognostic factor for both disease-specific and metastasis-free survival (P=0.026). It is concluded that strong tumour Bid expression is associated with poor outcome following radiotherapy regardless of intrinsic tumour cell radiosensitivity, and is adverse prognostic for disease-specific and metastasis-free survival in younger patients.
Bcl-2 family; cervix carcinoma; prognosis; metastasis; radiotherapy
The intracellular signals that mediate skeletal muscle protein loss and functional deficits due to muscular disuse are just beginning to be elucidated. Previously we showed that the activity of an NF-κB–dependent reporter gene was markedly increased in unloaded muscles, and p50 and Bcl-3 proteins were implicated in this induction. In the present study, mice with a knockout of the p105/p50 (Nfkb1) gene are shown to be resistant to the decrease in soleus fiber cross-sectional area that results from 10 days of hindlimb unloading. Furthermore, the marked unloading-induced activation of the NF-κB reporter gene in soleus muscles from WT mice was completely abolished in soleus muscles from Nfkb1 knockout mice. Knockout of the B cell lymphoma 3 (Bcl3) gene also showed an inhibition of fiber atrophy and an abolition of NF-κB reporter activity. With unloading, fast fibers from WT mice atrophied to a greater extent than slow fibers. Resistance to atrophy in both strains of knockout mice was demonstrated clearly in fast fibers, while slow fibers from only the Bcl3–/– mice showed atrophy inhibition. The slow-to-fast shift in myosin isoform expression due to unloading was also abolished in both Nfkb1 and Bcl3 knockout mice. Like the soleus muscles, plantaris muscles from Nfkb1–/– and Bcl3–/– mice also showed inhibition of atrophy with unloading. Thus both the Nfkb1 and the Bcl3 genes are necessary for unloading-induced atrophy and the associated phenotype transition.
The aim of the study was to evaluate VEGF expression in tumour biopsies as a prognostic factor for radiotherapy outcome in advanced carcinoma of the cervix. A retrospective study was carried out on 100 patients. Pre-treatment tumour VEGF expression was examined immunohistochemically in formalin-fixed, paraffin-embedded biopsies using a widely available commercial antibody. A semi-quantitative analysis was made using a scoring system of 0, 1, 2, and 3, for increasing intensity of staining. High VEGF expression was associated with a poor prognosis. A univariate log rank analysis found a significant relationship with overall survival (P = 0.0008) and metastasis-free survival (P = 0.0062), but not local control (P = 0.23). There was no correlation between VEGF expression and disease stage, tumour differentiation, patient age, or tumour radiosensitivity (SF2). In a Cox multivariate analysis of survival VEGF expression was the most significant independent prognostic factor (P = 0.001). After allowing for VEGF only SF2 was a significant prognostic factor (P = 0.003). In conclusion, immunohistochemical analysis of VEGF expression is a highly significant and independent prognostic indicator of overall and metastasis-free survival for patients treated with radiotherapy for advanced carcinoma of the cervix. It is also a rapid and easy method that could be used in the clinical setting, to identify patients at high risk of failure with conventional radiotherapy who may benefit from novel approaches or chemoradiotherapy. © 2000 Cancer Research Campaign
cervical carcinoma; vascular endothelial growth factor (VEGF); immunohistochemistry; prognosis
A relationship between hypoxia and apoptosis has been identified in vitro and in experimental tumours. The aim of this study was to investigate the relationship between apoptosis, hypoxia and the change in oxygenation during radiotherapy in human squamous cell carcinoma of the cervix. Forty-two patients with locally advanced disease underwent pretreatment evaluation of tumour oxygenation using an Eppendorf computerized microneedle electrode. Twenty-two of these patients also had a second evaluation of tumour oxygenation after receiving 40–45 Gy external beam radiotherapy. Paraffin-embedded histological sections were obtained from random pretreatment biopsies for all 42 patients. Apoptotic index (AI) was quantified by morphology on TUNEL stained sections. No correlation was found between pretreatment measures of AI and either the median pO2(r = 0.12, P = 0.44) or percentage of values < 5 mmHg (r = –0.02, P = 0.89). A significant positive correlation was found between AI and the change in tumour oxygenation (ratio of pre:post-treatment % values < 5 mmHg) following radiotherapy (r = 0.61, P = 0.002). The lack of correlation between apoptosis and hypoxia may occur because the Eppendorf measures both acute and chronic hypoxia, and the relative ability of acute hypoxia to induce apoptosis is unknown. These results indicate that cell death via apoptosis may be a mechanism of tumour reoxygenation during radiotherapy. © 2000 Cancer Research Campaign
cervix carcinoma; apoptosis; hypoxia; reoxygenation; radiotherapy
The purpose of this work was to compare the detection accuracy of 3-dimensional (3D) modalities of tuned-aperture computed tomography (TACT) with that of conventional 2-dimensional (2D) digital spot mammograms. A standardized mammographic phantom was placed beneath cadaveric breast tissues of varying densities. Five radiologists were asked to detect as many objects (specks, fibers, and lowcontrast masses) as possible from 90 displays in a controlled and factorially balanced multivariate experiment. Radiographic exposure was varied systematically, and projections were averaged to ensure stochastic comparability. Scores were weighted to eliminate task-specific bias and were analyzed by multivariate analyses of variance. All display modalities based on the linear application of the 3D TACT reconstruction method yielded significantly higher detection scores for all tasks than did conventional 2D digital spot mammography, which served as the scientific control modality. This effect was found to be statistically significant (P<.001) in spite of significant variations between tissues (P<.001), observers (P<.001), and exposures (P<.01). TACT may be a promising alternative or enhancement to conventional 2D digital mammography for tasks well simulated by this experimental design.
computed tomography; three-dimensional; observer performance; digital radiography; breast radiography; tomosynthesis
The aim of the study was to investigate the relationship between intrinsic radiosensitivity and vascularity in carcinoma of the cervix given radiotherapy, and assess whether more refined prognostic information can be gained by combining the two parameters. A retrospective study was carried out on 74 patients with locally advanced carcinoma of the cervix. Formalin-fixed, paraffin-embedded tumour biopsies were stained with anti-factor VIII using immunohistochemistry. Vascularity was scored using the intra-tumour microvessel density (IMD), or ‘hot-spot’, technique. For the same patients, the measurement of intrinsic radiosensitivity (SF2) had been made previously on the same pretherapy samples. Patients were stratified by the median IMD and SF2 scores. Women with radioresistant and highly vascular tumours had poorer 5-year survival (P = 0.0005, P = 0.035 respectively) and local control (P = 0.012, P = 0.077 respectively) than those with radiosensitive and poorly vascular tumours. No significant correlation was seen between IMD and SF2. Multivariate analysis (including tumour stage and patient age) showed that only SF2 and IMD were significant prognostic factors for survival. Patients with both a radioresistant and highly vascular tumour had a 5-year survival level of 18% compared to 77% for those patients with a radiosensitive and poorly vascularized tumour. Tumour angiogenesis and cellular radiosensitivity are independent prognostic factors for cervix carcinoma treated with radiotherapy. Allowing for tumour radiosensitivity increases the prognostic significance of vascularity measurements in cervix tumours. © 1999 Cancer Research Campaign
angiogenesis; SF2; predictive assays
As part of our programme for developing predictive tests for normal tissue response to radiotherapy, we have investigated the efficacy of the cytokinesis-block micronucleus (MN) assay as a means of detecting interindividual differences in cellular radiosensitivity. A study was made of nine fibroblast strains established from vaginal biopsies of pretreatment cervical cancer patients and an ataxia telangiectasia (A-T) cell strain. Cells were irradiated in plateau phase, replated and treated with cytochalasin B 24 h later. MN formation was examined 72 h after irradiation as the number of MN in 100 binucleate cells. The method yielded low spontaneous MN yields (<7 per 100 cells), and mean induced MN frequencies after 3.5 Gy varied between cell strains from 18 to 144 per 100 cells. However, in repeat experiments, considerable intrastrain variability was observed (CV = 32%), with up to twofold differences in MN yields, although this was less than interstrain variability (CV = 62%). An analysis was made of the relationship between MN results and previously obtained clonogenic survival data. There was a significant correlation between MN yields and clonogenic survival. However, when the A-T strain was excluded from the analysis, the correlation lost significance, mainly because of one slow-growing strain which was the most sensitive to cell killing but had almost the lowest MN frequency. With current methodology, the MN assay on human fibroblasts does not appear to have a role in predictive testing of normal tissue radiosensitivity.
A study was made of the relationship between the intrinsic radiosensitivity of human cervical tumours and the expression of the DNA repair enzyme human apurinic/apyrimidinic endonuclease (HAP1). The radiosensitivity of clonogenic cells in tumour biopsies was measured as surviving fraction at 2 Gy (SF2) using a soft agar assay. HAP1 expression levels were determined after staining of formalin-fixed paraffin-embedded tumour sections with a rabbit antiserum raised against recombinant HAP1. Both measurements were obtained on pretreatment biopsy material. All 25 tumours examined showed positive staining for HAP1, but there was heterogeneity in the level of expression both within and between tumours. The average coefficients of variation for intra- and intertumour heterogeneity were 62% and 82% respectively. There was a moderate but significant positive correlation between the levels of HAP1 expression and SF2 (r = 0.60, P = 0.002). Hence, this study shows that there is some relationship between intrinsic radiosensitivity and expression of a DNA repair enzyme in cervical carcinomas. The results suggest that this type of approach may be useful in the development of rapid predictive tests of tumour radiosensitivity.