Molecular characteristics of cancer vary between individuals. In future, most trials will require assessment of biomarkers to allocate patients into enriched populations in which targeted therapies are more likely to be effective. The MRC FOCUS3 trial is a feasibility study to assess key elements in the planning of such studies.
Patients and Methods:
Patients with advanced colorectal cancer were registered from 24 centres between February 2010 and April 2011. With their consent, patients' tumour samples were analysed for KRAS/BRAF oncogene mutation status and topoisomerase 1 (topo-1) immunohistochemistry. Patients were then classified into one of four molecular strata; within each strata patients were randomised to one of two hypothesis-driven experimental therapies or a common control arm (FOLFIRI chemotherapy). A 4-stage suite of patient information sheets (PISs) was developed to avoid patient overload.
A total of 332 patients were registered, 244 randomised. Among randomised patients, biomarker results were provided within 10 working days (w.d.) in 71%, 15 w.d. in 91% and 20 w.d. in 99%. DNA mutation analysis was 100% concordant between two laboratories. Over 90% of participants reported excellent understanding of all aspects of the trial. In this randomised phase II setting, omission of irinotecan in the low topo-1 group was associated with increased response rate and addition of cetuximab in the KRAS, BRAF wild-type cohort was associated with longer progression-free survival.
Patient samples can be collected and analysed within workable time frames and with reproducible mutation results. Complex multi-arm designs are acceptable to patients with good PIS. Randomisation within each cohort provides outcome data that can inform clinical practice.
colorectal cancer; biomarkers; multi-arm trials; personalised medicine
The interaction between ethanol (EtOH) and anxiety plays an integral role in the development and maintenance of alcoholism. Many medications in pre-clinical or clinical trials for the treatment of alcoholism share anxiolytic properties. However, these drugs typically have untoward side effects, such as sedation or impairment of motor function that may limit their clinical use. We have recently demonstrated that BRL 37344 (BRL), a selective β3-adrenoceptor agonist, enhances a discrete population of GABAergic synapses in the basolateral amygdala (BLA) that mediates feed-forward inhibition from lateral paracapsular (LPC) GABAergic interneurons onto BLA pyramidal cells. Behavioral studies revealed that intra-BLA infusion of BRL significantly reduced measures of unconditioned anxiety-like behavior without locomotor depressant effects.
The present studies tested the effect of BRL (0.1, 0.5, or 1.0μg/side) on EtOH self-administration using an intermittent access (IA) home cage two-bottle choice procedure, and limited access operant responding for EtOH or sucrose.
Intra-BLA infusion of BRL did not reduce homecage, intermittent EtOH self-administration. However, using an operant procedure that permits the discrete assessment of appetitive (seeking) and consummatory measures of EtOH self-administration, BRL reduced measures of EtOH and sucrose seeking, but selectively reduced operant responding for EtOH during extinction probe trials. BRL had no effect on consummatory behaviors for EtOH or sucrose.
Together, these data suggest that intra-BLA infusion of BRL significantly reduces motivation to seek EtOH and provide initial evidence that β3-ARs and LPC GABAergic synapses may represent promising targets for the development of novel pharmacotherapies for the treatment of alcoholism.
alcoholism; anxiety; GABA; negative reinforcement; norepinephrine
Mycobacterium tuberculosis infection generates pulmonary granulomas that consist of a caseous, necrotic core surrounded by an ordered arrangement of macrophages, neutrophils and T cells. This inflammatory pathology is essential for disease transmission and M. tuberculosis has evolved to stimulate inflammatory granuloma development while simultaneously avoiding destruction by the attracted phagocytes. The most abundant phagocyte in active necrotic granulomas is the neutrophil. Here we show that the ESAT-6 protein secreted by the ESX-1 type VII secretion system causes necrosis of the neutrophils. ESAT-6 induced an intracellular Ca2+ overload followed by necrosis of phosphatidylserine externalised neutrophils. This necrosis was dependent upon the Ca2+ activated protease calpain, as pharmacologic inhibition prevented this secondary necrosis. We also observed that the ESAT-6 induced increase in intracellular Ca2+, stimulated the production of neutrophil extracellular traps characterised by extruded DNA and myeloperoxidase. Thus we conclude that ESAT-6 has a leukocidin function, which may facilitate bacterial avoidance of the antimicrobial action of the neutrophil while contributing to the maintenance of inflammation and necrotic pathology necessary for granuloma formation and TB transmission.
Although alcoholism is a worldwide problem resulting in millions of deaths, only a small percentage of alcohol users become addicted. Notably, the specific neural substrates responsible for individual differences in vulnerability to alcohol addiction are not known. In these studies, we used rodent models to study behavioral and synaptic correlates related to individual differences in the development of ethanol locomotor sensitization, a form of drug-dependent behavioral plasticity associated with addiction vulnerability. Male Swiss mice were treated daily with saline or 1.8 g/kg ethanol for 21 days. Locomotor activity tests were performed once a week for 15 min immediately after saline or ethanol injections. After at least eleven days of withdrawal, cohorts of saline and ethanol-treated mice were used to characterize the relationships between locomotor sensitization, ethanol drinking, and glutamatergic synaptic transmission in the nucleus accumbens. Ethanol-treated mice that expressed locomotor behavioral sensitization to ethanol drank significantly more ethanol than saline-treated subjects and ethanol-treated animals resilient to this form of behavioral plasticity. Moreover, ethanolsensitized mice also had reduced accumbal NMDA receptor function and expression, as well as deficits in NMDA receptor-dependent long term depression in the nucleus accumbens core after a protracted withdrawal. These findings suggest that disruption of accumbal core NMDA receptor-dependent plasticity may represent a synaptic correlate associated with ethanol-induced locomotor sensitization and increased propensity to consume ethanol.
Long-Term Depression; Nucleus Accumbens; Behavioral sensitization; Ethanol; Voluntary drinking; Glutamate receptors
One of the factors influencing the cost-effectiveness of population screening for type 2 diabetes may be uptake. We examined attendance and practice- and individual-level factors influencing uptake at each stage of a diabetes screening programme in general practice.
A stepwise screening programme was undertaken among 135,825 people aged 40-69 years without known diabetes in 49 general practices in East England. The programme included a score based on routinely available data (age, sex, BMI and prescribed medication) to identify those at high risk who were offered random capillary blood glucose (RBG) and glycosylated haemoglobin tests. Those screening positive were offered fasting capillary blood glucose (FBG) and confirmatory oral glucose tolerance tests (OGTT).
33,539 high risk individuals were invited for a RBG screening test; 24,654 (74%) attended. 94% attended the follow-up FBG test and 82% the diagnostic OGTT. 70% of individuals completed the screening programme. Practices with higher GP staff complements and those located in more deprived areas had lower uptake for RBG and FBG tests. Male sex and a higher BMI were associated with lower attendance for RBG testing. Older age, prescription of antihypertensive medication and a higher risk score were associated with higher attendance for FBG and RBG tests.
High attendance rates can be achieved by targeted stepwise screening of individuals assessed as high risk by data routinely available in general practice. Different strategies may be required to increase initial attendance, ensure completion of the screening programme, and reduce the risk that screening increases health inequalities.
Screening; type 2 diabetes; programme; population; ADDITION
Most studies of total hip arthroplasty (THA) focus on the effect of the type of implant on the clinical result. Relatively little data are available on the impact of the patient’s preoperative status and socioeconomic factors on the clinical results following THA.
We determined the relative importance of patient preoperative and socioeconomic status compared to implant and technique factors in predicting patient outcome as reflected by scores on commonly utilized rating scales (eg, Harris Hip Score, WOMAC, SF-12, degree of patient satisfaction, or presence or severity of thigh pain) following cementless THA.
All patients during the study period were offered enrollment in a prospective, randomized study to receive either a titanium, tapered, proximally coated stem; or a Co-Cr, cylindrical, extensively coated stem; 102 patients were enrolled. We collected detailed patient data preoperatively including diagnosis, age, gender, insurance status, medical comorbidities, tobacco and alcohol use, household income, educational level, and history of treatment for lumbar spine pathology. Clinical evaluation included Harris Hip Score, SF-12, WOMAC, pain drawing, and UCLA activity rating and satisfaction questionnaire. Implant factors included stem type, stem size, fit in the canal, and stem-bone stiffness ratios. Minimum 2 year followup was obtained in 95% of the enrolled patients (102 patients).
Patient demographics and preoperative status were more important than implant factors in predicting the presence of thigh pain, dissatisfaction, and a low hip score. The most predictive factors were ethnicity, educational level, poverty level, income, and a low preoperative WOMAC score or preoperative SF-12 mental component score. No implant parameter correlated with outcome or satisfaction.
Socioeconomic factors and preoperative status have more impact on the clinical outcome of cementless THA than implant related factors.
Level of Evidence
Level I, prospective, randomized clinical trial. See the guidelines online for a complete description of level of evidence.
A combined laboratory and modeling approach was used to assess the impact of selected pesticides on early life stages of the soft-shell clam, Mya arenaria. Clams were exposed for 24 h as veligers or pediveligers to the broad-spectrum herbicide hexazinone [3-cyclohexyl-6-(dimethylamino)-1-methyl-1,3,5-triazine-2,4 (1h,3h)-dione; (Velpar®)], the phenoxyacetic acid herbicide, 2,4-D (2,4- dichlorophenoxyacetic acid; Agway® Super BK 32), or phosmet (Imidan®). In addition, juvenile clams were exposed for 24 h to 2,4-D and their growth monitored for 21 months. Laboratory experiments indicated veligers were more sensitive to acute pesticide exposure than pediveligers, with 2,4-D exposed veligers exhibiting the lowest survival among all treatments. Relative to controls, juvenile clams exposed to 0.5 ppm 2,4-D had enhanced survival following the initial 3 months of grow out. Juveniles exposed to 0.5 ppm, 5 ppm and 10 ppm 2,4-D showed an initial growth delay relative to control clams, but at 21 months post exposure these clams were significantly larger than control clams. Data from the larval and juvenile exposures were used to generate a stage-specific matrix model to predict the effect of pesticide exposure on clam populations. Impacts on simulated clam populations varied with the pesticide and stage exposed. For example, 2,4-D exposure of veligers and pediveligers significantly reduced predicted recruitment as well as population growth rate compared to controls, but juvenile exposure to 2,4-D did not significantly reduce population growth rate. With the exception of veligers exposed to 10 ppm, hexazinone exposure at the both veliger and pediveliger stages significantly reduced predicted recruitment success compared to 0 ppm controls. Hexazinone exposure also reduced modeled population growth rates, but these reductions were only slight in the pediveliger exposure simulations. Veliger and pediveliger exposure to phosmet reduced modeled population growth rate in a dose-dependent fashion. Changes in modeled population stable stage distributions were also observed when veligers were exposed to any pesticide. These results suggest that both the stage of exposure and the specific toxicant are important in predicting effects of pesticide exposure on soft-shell clam populations, with earlier life stages showing greater sensitivity to the pesticides tested.
bivalve; 2,4-D; hexazinone; phosmet; pediveliger; veliger; matrix population model
MECP2 mutations are identifiable in ∼80% of classic Rett syndrome (RTT), but less frequently in atypical RTT. We recruited 110 patients who fulfilled the diagnostic criteria for Rett syndrome and were referred to Cardiff for molecular analysis, but in whom an MECP2 mutation was not identifiable. Dosage analysis of MECP2 was carried out using multiplex ligation dependent probe amplification or quantitative fluorescent PCR. Large deletions were identified in 37.8% (14/37) of classic and 7.5% (4/53) of atypical RTT patients. Most large deletions contained a breakpoint in the deletion prone region of exon 4. The clinical phenotype was ascertained in all 18 of the deleted cases and in four further cases with large deletions identified in Goettingen. Five patients with large deletions had additional congenital anomalies, which was significantly more than in RTT patients with other MECP2 mutations (2/193; p<0.0001). Quantitative analysis should be included in molecular diagnostic strategies in both classic and atypical RTT.
Gross gene deletion; MECP2; Rett syndrome
Aims: Members of the acid sensing ion channel (ASIC) family are strong candidates as mechanical transducers in sensory function. The authors have shown that ASIC1a has no role in skin but a clear influence in gastrointestinal mechanotransduction. Here they investigate further ASIC1a in gut mechanoreceptors, and compare its influence with ASIC2 and ASIC3.
Methods and results: Expression of ASIC1a, 2, and 3 mRNA was found in vagal (nodose) and dorsal root ganglia (DRG), and was lost in mice lacking the respective genes. Recordings of different classes of splanchnic colonic afferents and vagal gastro-oesophageal afferents revealed that disruption of ASIC1a increased the mechanical sensitivity of all afferents in both locations. Disruption of ASIC2 had varied effects: increased mechanosensitivity in gastro-oesophageal mucosal endings, decreases in gastro-oesophageal tension receptors, increases in colonic serosal endings, and no change in colonic mesenteric endings. In ASIC3-/- mice, all afferent classes had markedly reduced mechanosensitivity except gastro-oesophageal mucosal receptors. Observations of gastric emptying and faecal output confirmed that increases in mechanosensitivity translate to changes in digestive function in conscious animals.
Conclusions: These data show that ASIC3 makes a critical positive contribution to mechanosensitivity in three out of four classes of visceral afferents. The presence of ASIC1a appears to provide an inhibitory contribution to the ion channel complex, whereas the role of ASIC2 differs widely across subclasses of afferents. These findings contrast sharply with the effects of ASIC1, 2, and 3 in skin, suggesting that targeting these subunits with pharmacological agents may have different and more pronounced effects on mechanosensitivity in the viscera.
ASIC channel; visceral afferents; splanchnic nerves; vagus nerves; knockout mice
Background: Gastric emptying is frequently delayed in critical illness which compromises the success of nasogastric nutrition. The underlying motor dysfunctions are poorly defined.
Aims: To characterise antro-pyloro-duodenal motility during fasting, and in response to gastric and duodenal nutrient, as well as to evaluate the relationship between gastric emptying and motility, in the critically ill.
Subjects: Fifteen mechanically ventilated patients from a mixed intensive care unit; 10 healthy volunteers.
Methods: Antro-pyloro-duodenal pressures were recorded during fasting, after intragastric administration (100 ml; 100 kcal), and during small intestinal infusion of liquid nutrient (6 hours; 1 kcal/min). Gastric emptying was measured using a 13C octanoate breath test.
Results: In healthy subjects, neither gastric nor small intestinal nutrient affected antro-pyloro-duodenal pressures. In patients, duodenal nutrient infusion reduced antral activity compared with both fasting and healthy subjects (0.03 (0–2.47) waves/min v 0.14 (0–2.2) fasting (p = 0.016); and v 0.33 (0–2.57)/min in healthy subjects (p = 0.005)). Basal pyloric pressure and the frequency of phasic pyloric pressure waves were increased in patients during duodenal nutrient infusion (3.12 (1.06) mm Hg; 0.98 (0.13)/min) compared with healthy subjects (−0.44 (1.25) mm Hg; p<0.02 after 120 minutes; 0.29 (0.15)/min; p = 0.0002) and with fasting (−0.06 (1.05) mm Hg; p<0.03 after 160 minutes; 0.49 (0.13)/min; (p = 0.0001). Gastric emptying was delayed in patients (gastric emptying coefficient 2.99 (0.2) v 3.47 (0.1); p = 0.015) and inversely related to the number of pyloric pressure waves (r = −0.563, p = 0.029).
Conclusions: Stimulation of pyloric and suppression of antral pressures by duodenal nutrient are enhanced in the critically ill and related to decreased gastric emptying.
manometry; gastrointestinal motility; pylorus; critical illness; gastric emptying
Prolonged exposure to organophosphate (OP) pesticides may produce cognitive deficits reflective of hippocampal injury in both humans and rodents. Recent work has indicated that microtubule trafficking is also adversely affected by exposure to the OP pesticide chlorpyrifos, suggesting a novel mode of OP-induced neurotoxicity. The present studies examined effects of prolonged exposure to chlorpyrifos-oxon (CPO) on acetylcholinesterase (AChE) activity, immunoreactivity (IR) of microtubule-associated proteins, neuronal injury, and tubulin polymerization using in vitro organotypic slice cultures of rat hippocampus and bovine tubulin. Cultures were exposed to CPO (0.1-10 μM) in cell culture medium for 1-7 days, a regimen producing progressive reductions in AChE activity of 15-60%. Cytotoxicity (somatic uptake of the non-vital marker propidium iodide, as well as, IR of α-tubulin and microtubule-associated protein-2 (a/b) [MAP-2] were assessed 1, 3, and 7 days after the start of CPO exposure. As early as 24- hr after the start of exposure, CPO-induced deficits in MAP-2 IR were evident and progressive in each region of slice cultures at concentrations as low as 0.1 μM. CPO exposure did not alter α-tubulin IR at any time point. Concentration-dependent injury in the CA1 pyramidal cell layer and to a lesser extent, CA3 and dentate cells, was evident 3 days after the start of CPO exposure (≥ 0.1 μM) and was greatest after 7 days. Tubulin polymerization assays indicated that CPO (≥ 0.1 μM) markedly inhibited the polymerization of purified tubulin and map-rich tubulin, though effects on MAP-rich tubulin were more pronounced. These data suggest that exposure to CPO produces a progressive decrease in neuronal viability that may be associated with impaired microtubule synthesis and/or function.
coronary dissection; coronary arteries
This paper presents the first full micro costing of a commonly used cancer genetic counselling and testing protocol used in the UK. Costs were estimated for the Cardiff clinic of the Cancer Genetics Service in Wales by issuing a questionnaire to all staff, conducting an audit of clinic rooms and equipment and obtaining gross unit costs from the finance department. A total of 22 distinct event pathways were identified for patients at risk of developing breast, ovarian, breast and ovarian or colorectal cancer. The mean cost per patient were £97–£151 for patients at moderate risk, £975–£3072 for patients at high risk of developing colorectal cancer and £675–£2909 for patients at high risk of developing breast or ovarian cancer. The most expensive element of cancer genetic services was labour. Labour costs were dependent upon the amount of labour, staff grade, number of counsellors used and the proportion of staff time devoted to indirect patient contact. With the growing demand for cancer genetic services and the growing number of national and regional cancer genetic centers, there is a need for the different protocols being used to be thoroughly evaluated in terms of costs and outcomes.
genetic; breast; ovarian; colorectal; cost
Background: Antropyloric motility is important for regulation of gastric emptying and has not been adequately characterised in premature infants.
Aim: To evaluate fed patterns of antropyloric motility in premature infants.
Subjects: Forty three healthy premature infants, 30–38 weeks of postmenstrual age.
Methods: Postprandial antropyloric motility was measured using a micromanometric feeding assembly (outer diameter 1.8 mm) incorporating a pyloric sleeve sensor. The occurrence of isolated pyloric pressure waves (IPPWs) and antral pressure wave sequences (PWSs) was characterised. Sequences were further classified as being antegrade, synchronous, antegrade-synchronous, and retrograde according to the direction of propagation.
Results: A total of 7289 pressure wave events were recorded, 48% IPPWs and 52% PWSs (18% antegrade, 12% synchronous, 13% antegrade-synchronous, 2% retrograde, and 7% undefined). IPPWs predominated in the first postprandial hour, peaking at 30–60 minutes. PWSs predominated in the period after one hour postprandially. Mean (SEM) half gastric emptying time was 42 (4) minutes.
Conclusions: Monitoring of antropyloric motor patterns in healthy premature infants indicates that the neuroregulatory mechanisms responsible for the coordination of antropyloric motility and gastric emptying are well developed by 30 weeks of postmenstrual age.
ReoPro (abciximab) is an extremely potent inhibitor of the glycoprotein IIb/IIIa receptor, the final common pathway of platelet activation and aggregation. Its main role is the maintenance of coronary patency after suboptimal results with coronary intervention. However, one of the complications of this treatment is excessive bleeding, a problem which may be compounded by a rare idiosyncratic thrombocytopenic reaction. A severe episode of thrombocytopenia in a 64 year old man is described; he was treated with ReoPro for a right coronary stenosis which had not been resolved by angioplasty. His platelet level dropped quickly and only improved after 20 units of platelets were given.
Keywords: ReoPro; abciximab; platelets; thrombocytopenia; interventional cardiology
Exogenous administration of 5-aminolevulinic acid (ALA) is becoming widely used to enhance the endogenous synthesis of Protoporphyrin IX (PpIX) in photodynamic therapy. We analysed porphyrin formation in chemically induced squamous papillomas, after topical application of ALA and ALA hexyl ester (He-ALA) administered in different formulations, as well as the pattern of distribution in the internal organs, and the synthesis of porphyrins in distant tumoural and normal skins. A lotion formulation containing DMSO and ethanol was the best vehicle for topical ALA delivery to papillomas, whereas cream was the most efficient formulation for He-ALA application. Similar porphyrin concentration can be accumulated in the skin tumours employing either ALA or He-ALA delivered in their optimal formulations. The use of cream as a vehicle of both ALA and He-ALA, induces highest porphyrin tumour/normal skin ratios. The main advantage of using He-ALA is that porphyrins synthesized from the ester are more confined to the site of application, thus inducing low porphyrin levels in normal skin, liver, blood and spleen, as well as in papillomas distant from the point of application, independently on the vehicle employed, so reducing potential side effects of photodynamic therapy. © 2001 Cancer Research Campaign http://www.bjcancer.com
ALA; ALA esters; photodynamic therapy; squamous papillomas
Smoking is a major risk factor for developing atherosclerosis. In order to understand the vascular abnormalities observed in smokers, we investigated vascular responsiveness in cigarette smokers.
We performed two consecutive matched group comparative studies to investigate vascular responsiveness using venous occlusion plethysmography. The mean effects of three incremental doses of each vasoactive agent are presented. Both studies compared smokers with nonsmokers.
The first investigated 68 subjects (smokers = 29; mean ± s.d. ages; 24 ± 6 vs 25 ± 5 years; P = NS) and found smoking was associated with a significant blunting of the flow ratio between treated and untreated arms to endothelium-dependent vasodilatation to acetylcholine (mean ± s.d., nonsmokers vs smokers) 4.07 ± 2.18 vs 3.42 ± 1.79 (P = 0.04, 95% CI 0.02, 1.12). By contrast, there was no significant difference in the responses to the endothelium-independent vasodilators sodium nitroprusside and verapamil. Smoking was also associated with a significant impairment in endothelium-dependent vasoconstriction induced by monomethyl-l-arginine (L-NMMA) 0.78 ± 0.22 vs 0.87 ± 0.21 (P = 0.006, 95% CI −0.14, −0.02) and a trend to blunted endothelium-independent vasoconstrictor responses to noradrenaline. In the second study we investigated the response to angiotensin I and II in 23 subjects (smokers = 12; mean ± s.d. ages; 34 ± 10 vs 32 ± 11 years). There was significant impairment in smokers of the mean vasoconstrictor response to angiotensin I 0.51 ± 0.15 vs 0.59 ± 0.16 (nonsmokers vs smokers; P = 0.003, 95% CI −0.13, −0.03) and a nonsignificant trend towards impairment of the response to angiotensin II.
Cigarette smoking in male volunteers is associated with blunted basal and stimulated nitric oxide bioactivity. Endothelial independent vasodilator responses (to nitroprusside and verapamil) were unaltered in smokers. A defect in the vasoconstrictor response to angiotensin I was also seen.
angiotensin I; endothelium; nitric oxide; smoking
the relation between nocturnal vasopressin release and response to
treatment with the vasopressin analogue
1-desamino-8-D-arginine vasopressin (DDAVP) in children
with primary monosymptomatic nocturnal enuresis.
recruited from a specific enuresis clinic and entered into a defined
treatment programme. Nocturnal vasopressin concentrations were measured
every 15 minutes over a four hour period during overnight admission.
children were eligible for entry into the study, 35 of whom agreed to
overnight sampling. There was a quadratic relation between mean plasma
AVP and response to treatment with DDAVP, with very high or very low
concentrations being unresponsive. Plasma AVP profiles ranged from low
concentrations with little variability to high concentrations with wide variability.
to respond to DDAVP is related to endogenous AVP production and is
influenced by neuronal patterning in early infancy. The best predictors
of success with treatment were a past history of breast feeding, mean
nocturnal AVP concentration, and the height of the child. The response
was adversely affected by poor weight at birth and poor linear growth.
The study suggests differing causes of nocturnal enuresis related to
different patterns of AVP release.
Samples of human and rat skin in short-term organ culture exposed to ALA or a range of hydrophobic derivatives were examined for their effect on the accumulation of protoporphyrin IX (PpIX) measured using fluorescence spectroscopy. With the exception of carbobenzoyloxy-D-phenylalanyl-5-ALA-ethyl ester the data presented indicate that, in normal tissues, ALA derivatives generate protoporphyrin IX more slowly than ALA, suggesting that they are less rapidly taken up and/or converted to free ALA. However, the resultant depot effect may lead to the enhanced accumulation of porphyrin over long exposure periods, particularly in the case of ALA-methyl ester or ALA-hexyl ester, depending on the applied concentration and the exposed tissue. Addition of the iron chelator, CP94, greatly increased PpIX accumulation in human skin exposed to ALA, ALA-methyl ester and ALA-hexyl ester. The effect in rat skin was less marked. © 1999 Cancer Research Campaign
ALA; PDT; ALA derivatives; ALA esters; iron chelators; CP94
Previous studies show that linking acetylated glucosamine to S-nitroso-N-acetyl-D,L-penicillamine (SNAP) stabilizes the molecule and causes it to elicit unusually prolonged vasodilator effects in endothelium-denuded, isolated rat femoral arteries. Here we studied the propanoyl (SNPP; 3 carbon side-chain), valeryl (SNVP; 5C) and heptanoyl (SNHP; 7C) N-substituted analogues of SNAP (2C), to further investigate other molecular characteristics that might influence chemical stability and duration of vascular action of S-nitrosothiols.Spectrophotometric analysis revealed that SNVP was the most stable analogue in solution. Decomposition of all four compounds was accelerated by Cu(II) and cysteine, and neocuproine, a specific Cu(I) chelator, slowed decomposition of SNHP. Generation of NO from the compounds was confirmed by electrochemical detection at 37°C.Bolus injections of SNAP (10 μl; 10−8–10−3 M) into the perfusate of precontracted, isolated rat femoral arteries taken from adult male Wistar rats (400–500 g), caused concentration-dependent, transient vasodilatations irrespective of endothelial integrity. Equivalent vasodilatations induced by SNVP and SNHP were transient in endothelium-intact vessels but failed to recover to pre-injection pressures at moderate and high concentrations (10−6–10−3 M) in those denuded of endothelium. This sustained effect (>1 h) was most prevalent with SNHP and was largely reversed by the NO scavenger, haemoglobin.We suggest that increased lipophilicity of SNAP analogues with longer sidechains facilitates their retention by endothelium-denuded vessels; subsequent slow decomposition within the tissue generates sufficient NO to cause prolonged vasodilatation. This is a potentially useful characteristic for targeting NO delivery to areas of endothelial damage.
Nitric oxide; S-nitrosothiols; vasodilatation; SNAP analogues
BACKGROUND: The government encourages general practitioners (GPs) to become involved in caring for drug users. However, in some areas of the country, including Bedford, secondary care support is inadequate. GPs in these areas have to decide how to cope with such patients entirely within general practice. AIM: To assess the characteristics and quality of care given without secondary care support to drug users by one practice in Bedford over a decade. METHOD: A search was made of the practice computer for all patients with a problem title of 'addiction drug' between 1986 and 1995. The age, sex, social characteristics, and drug history were recorded. RESULTS: One hundred and ninety-two patients were found, of which 155 took part in the practice programme; i.e. they consulted more than three times. Forty-three patients (37%) who took part and were prescribed Methadone were prescribed this drug as ampoules. Sixty-three patients (40.6%) who took part in the programme stopped using drugs. Thirty-two (33.6%) of the Methadone users became abstinent. A higher proportion of women (13-48%) than men (19-27.7%) stopped using Methadone (P = 0.019). Among patients who had a stable lifestyle, a higher proportion had been prescribed ampoules than mixture (22 out of 28: 78.6%; P = 0.001). Similarly, of those who had a job, eight out of 11 (72%; P = 0.037) had been prescribed methadone ampoules. Two-thirds of all patients prescribed amphetamines stopped using drugs. CONCLUSION: Long-term care of drug users entirely within general practice is feasible. Among those prescribed methadone ampoules, a higher than average proportion had stable lifestyles and had a stable job.
OBJECTIVE--To identify antigen(s) among purified deglycosylated aggrecan peptides spanning the chondroitin sulphate domain that may be responsible for the initiation or perpetuation of the autoimmune responses in rheumatoid arthritis (RA). METHODS--Aggrecan was purified from human articular cartilage and deglycosylated with either bacterial glycosidases or trifluoromethanesulphonic acid (TFMS). Twelve overlapping peptides (15 residues) spanning the chondroitin sulphate domain with N-terminal residues offset by three amino acids were synthesised. T cell responses to these antigens in RA patients and age matched controls were assessed in vitro by antigen specific T cell proliferation assays. RESULTS--Enzymically deglycosylated aggrecan (EDA) stimulated proliferation of T cells isolated from the peripheral blood in a greater proportion of patients with RA than controls. In a subset (12.5%) of RA patients, the magnitude of stimulation lay outside the control range. T cell proliferative responses to TFMS treated aggrecan were greater than, but well correlated with, responses to EDA. T cells from 15 patients were also stimulated with the pooled synthetic peptides. Four of seven patients who demonstrated T cell reactivity to EDA (seven of 15) also showed enhanced T cell proliferation to synthetic peptides. CONCLUSION--These data suggest that an autoantigenic T cell epitope may lie within the chondroitin sulphate domain of aggrecan.
The vasodilator properties of a novel S-nitrosated glyco-amino acid (RIG200) were investigated in isolated rat femoral arteries and compared with those of the parent S-nitrosothiol compound, S-nitroso-N-acetylpenicillamine (SNAP).Spectrophotometric analysis revealed that 2.5 mM solutions of RIG200 decomposed more slowly (half-life (t1/2)=216.2±26.7 min) than SNAP (t1/2=37.2±13.8 min) in Krebs buffer at 24°C. Furthermore, the rate of decomposition of SNAP, but not of RIG200, was significantly reduced by the Cu(I) chelator, neocuproine. We concluded that the relative stability of RIG200 is due, at least in part, to its resistance to trace Cu(I)-catalyzed decomposition. Nitric oxide (NO) generation from SNAP and RIG200 was confirmed by use of an NO electrode.Experiments to investigate the vasodilator effects of RIG200 were carried out on isolated femoral arteries taken from adult male Wistar rats (400–550 g). Lengths of artery (7–8 mm long) were cannulated, dissected free and perfused at constant flow rate (0.6 ml min−1) with Krebs buffer. Vessels were precontracted with phenylephrine (10.2±0.3 μM) and developed pressures of 91.8 ± 4 mmHg, detected upstream by a differential pressure transducer.Concentration-dependent vasodilator responses to bolus injections of SNAP or RIG200 (10 μl; 10−8–10−3 M) made into the perfusate of endothelium-intact vessels were transient, recovering the pre-injection pressure in <20 min.Responses to equivalent bolus injections of SNAP in endothelium-denuded vessels were also transient but those in response to concentrations of RIG200 >10−5 M were sustained. Responses to 10−3 M RIG200 were sustained for periods >4 h. Sustained vasodilatation was reversed by the NO scavenger, ferrohaemoglobin (10 μM) but was unaffected by the NO synthase inhibitor, Nω-nitro-L-arginine methyl ester (200 μM), indicating involvement of NO from a source other than NO synthase.We suggest that a possible explanation for the prolonged effect of RIG200 is retention of the compound by the vascular wall, facilitated by endothelial denudation. Slow decomposition of RIG200 in situ would release sufficient NO to maintain a ‘vasodilator tone' which persists for more than 4 h. Selective retention by damaged vessels could have important therapeutic implications with regard to targeted delivery of NO, restoring protection to areas deprived of endogenous NO, whilst avoiding unwanted hypotension.
Nitric oxide; S-nitrosothiols; vasodilatation; RIG200