Elevated non-esterified fatty acids (NEFA) concentrations in non-pregnant animals have been reported to decrease pancreatic responsiveness. As ovine gestation advances, maternal insulin concentrations fall and NEFA concentrations increase. Experiments were designed to examine if the pregnancy-associated rise in NEFA concentration is associated with a reduced pancreatic sensitivity to glucose in vivo. We investigated the possible relationship of NEFA concentrations in regulating maternal insulin concentrations during ovine pregnancy at three physiological states, non-pregnant, non-lactating (NPNL), 105 and 135 days gestational age .t-tests, repeated measures ANOVA and 2-way ANOVA.The plasma concentrations of insulin, growth hormone (GH) and ovine placental lactogen (oPL) were determined by double antibody radioimmunoassay. Insulin responsiveness to glucose was measured using bolus injection and hyperglycaemic clamp techniques in 15 non-pregnant, non-lactating ewes and in nine pregnant ewes at 105 dGA and near term at 135 dGA. Plasma samples were also collected for hormone determination. In addition to bolus injection glucose and insulin Area Under Curve calculations, the Mean Plasma Glucose Increment, Glucose Infusion Rate and Mean Plasma Insulin Increment and Area Under Curve were determined for the hyperglycaemic clamp procedures. Statistical analysis of data was conducted with Students Maternal growth hormone, placental lactogen and NEFA concentrations increased, while basal glucose and insulin concentrations declined with advancing gestation. At 135 dGA following bolus glucose injections, peak insulin concentrations and insulin area under curve (AUC) profiles were significantly reduced in pregnant ewes compared with NPNL control ewes . In hyperglycaemic clamp studies, while maintaining glucose levels not different from NPNL ewes, pregnant ewes displayed significantly reduced insulin responses and a maintained depressed insulin secretion. In NPNL ewes, 105 and 135 dGA ewes, the Glucose Infusion Rate (GIR) was constant at approximately 5.8 mg glucose/kg/min during the last 40 minutes of the hyperglycaemic clamp and the Mean Plasma Insulin Increment (MPII) was only significantly (p < 0.001) greater in NPNL ewes. Following the clamp, NEFA concentrations were reduced by approximately 60% of pre-clamp levels in all groups, though a blunted and suppressed insulin response was maintained in 105 and 135 dGA ewes.Results suggest that despite an acute suppression of circulating NEFA concentrations during pregnancy, the associated steroids and hormones of pregnancy and possibly NEFA metabolism, may act to maintain a reduced insulin output, thereby sparing glucose for non-insulin dependent placental uptake and ultimately, fetal requirements. To meet the increasing fetal demands and maternal energy requirements of pregnancy, alterations in the partitioning and utilization of maternal nutrients must occur. These adaptations are regulated by changing blood concentrations of regulatory metabolites and hormones, together with changes in target tissue responsiveness. Of principle interest are alterations in maternal glucose metabolism during pregnancy, as glucose is a major limiting nutrient of fetal growth ,2. As shHigh plasma NEFA concentrations have been associated with the development of insulin resistance in the peripheral tissues and also the β-cell . Elevatein vivo. Reported here are in vivo pregnant sheep studies of insulin responsiveness to glucose, measured using bolus injection and hyperglycemic clamp techniques.Studies investigating a possible interaction between increasing NEFA concentrations and maternal pancreatic sensitivity during ovine pregnancy have not yet been reported. The following experiments were designed to examine whether the pregnancy-associated rise in NEFA concentration is associated with a reduced pancreatic sensitivity to glucose Twenty-four 3–4 year old, pen-trained Merino ewes of a known gestational age were used. The experiments were conducted adhering to the National Health and Medical Research Council (NH&MRC) guidelines as administered and approved by the University of Western Sydney, Hawkesbury Animal Care and Ethics Committee and the Commonwealth Scientific Research Organization, Division of Animal Production, Animal Care and Experimentation Ethics Committee. All animals displaying estrus following a program of synchronised mating induced by pre-treatment with intravaginal progestogen pessaries were recorded. Pregnancy was later verified by rectal ultrasound scanning at 28 dGA, and then confirmed together with litter size determination at 65 dGA, using abdominal ultrasound. Those ewes displaying estrus, but failing to conceive were used in a non-pregnant, non-lactating (NPNL) control group. A total of 15 NPNL ewes and nine pregnant ewes, three single bearing and six twin bearing, were used in this study. At 90 dGA a trial glucose bolus injection study was conducted in four ewes. At 105 dGA, all animals were studied , in both glucose bolus and hyperglycaemic protocols and then subsequently again at 135 dGA. At each gestational age, NPNL ewes (n = 15) were also studied under both protocols, expect at 90 dGA were only the bolus studies were conducted and only 5 NPNL animals studied.One week prior to the commencement of the experimental period, ewes were weighed. Body weights (BW) were used to determine the glucose bolus doses to be administered at study (0.4 g glucose/kg). Animals were then individually penned under natural lighting conditions and fed 700 g/d per animal, of a 60:40 pelleted ration, consisting of 60% hammer milled lucerne and 40% hammer milled oaten chaff (92.7 ± 0.34 % dry matter (DM), 68.9 ± 1.05% digestible organic matter, and having a calculated metabolizable energy content of 10.33 ± 0.2 MJ/kg DM, crude protein content of 13.8 ± 0.5%). Water was freely available at all times.The ewes were subjected to an experiment protocol consisting of two procedures, 1) a bolus glucose study and 2) a hyperglycaemic clamp study. Each experimental period extended over 5 days, with 2 days in between the procedures. On the day before the commencement of the bolus glucose study, each ewe had polyvinyl jugular vein catheters inserted bilaterally under local anaesthesia. Catheters were maintained patent with daily-heparinized saline flushes (35 U heparin/ml). The bolus glucose procedure was conducted as follows. On the day of the experiment, following a 24-h fast, two pre-injection  blood samples were collected 15 min apart. Immediately following the second basal sample, a glucose bolus was administered. Seven two ml blood samples were collected at 5, 10, 20, 35, 55, 155 and 215 min post-injection and stored in an ice bath until centrifuged at 1,200 × g for 15 min at 4°C. A 0.5 ml aliquot of supernatant was removed for glucose determination, while the remaining plasma was frozen at -20°C for later biochemical analysis.® blood glucose monitoring kit (Boehringer Germany). The pump speed setting was then adjusted to maintain the required blood glucose concentration. Samples were collected for analysis as described for the bolus protocol.Two days after the bolus glucose injection study, animals were also subjected to a 120 min hyperglycaemic clamp procedure, which was imposed after a 24 h fast . Prior tInsulin; Inter-assay LQC, 5.2, HQC, 3.9 and intra-assay LQC, 5.7, HQC, 7.3. GH; Inter-assay LQC 10.3, HQC 7.9, and intra-assay LQC 8.6, HQC 7.5.oPL; Inter-assay LQC 9.1, HQC 12.0, and intra-assay LQC 9.2, HQC 10.9. Glucose; Inter-assay LQC 10.1, HQC 9.3, and intra-assay LQC 10.3, HQC 11.9. NEFA; Inter-assay LQC 11.4, HQC 7.8, and intra-assay LQC 10.6, 6.7.The plasma concentrations of insulin, growth hormone (GH) and ovine placental lactogen (oPL) were determined by double antibody radioimmunoassay as previously described . InsulinData from the bolus glucose injection studies were expressed using the following parameters;- Area under curve (AUC), for both the glucose ((mg/ml)/min) and insulin ((ng/ml)/min) response profiles,- Glucose clearance rate - Glucose half life AUC for both the glucose and the resulting insulin response profiles were calculated using the trapezoidal rule. Glucose clearance rate  was calculated as Glucose dose /AUC. Glucose and insulin half-life was determined from the interpolated cumulative response curve. Glucose and insulin concentrations at 10 minutes were used as the peak response. In the hyperglycaemic clamp studies, in addition to AUC calculations for the whole study period, the Mean Plasma Glucose Increment , Glucose Infusion Rate /kg BW/min) and Mean Plasma Insulin Increment  were determined over the last 40 min of the clamp , during t-tests. Changes in NEFA concentrations following hyperglycaemic clamp treatment at three different physiological states, NPNL, 105 dGA and 135 dGA was analysed using 2 way ANOVA.There was no significant time of experiment effect when comparing NPNL ewe data collected at each of the gestational age studied, and as a consequence NPNL data from studies at 90, 105 and 135 dGA were pooled and analysed as a single control group against each gestational age group. No differences were found between the singleton and twin bearing ewes and litter size data were pooled to give one pregnant animal group at 105 and 135 dGA (n = 9). Glucose data from the 90, 105 and 135 dGA bolus glucose and 105 and 135 dGA hyperglycaemic clamp experiments were analysed through repeated measures ANOVA with Greenhouse Geisser adjustment . BetweenThere were no significant differences in body weight (Mean weight: 48.4 ± 1.8 kgs) and required glucose bolus injection (19.5 ± 0.7 g) across the physiological states studied. In the bolus glucose experiments, the basal glucose concentration was significantly lower in pregnant ewes measured on day 135 than on day 90 of gestation (p < 0.05). Basal glucose concentrations in the clamp studies, at 105 and 135 dGA, were also significantly reduced compared to NPNL concentrations , to 2.13 ± 0.35 ng/ml at 105 dGA and 3.5 ± 0.9 ng/ml at 135 dGA. Placental lactogen concentrations were 579 ± 74 ng/ml at 105 dGA and increased to 1211 ± 144 ng/ml (p < 0.05) by 135 dGA. Both pregnant groups displayed higher NEFA concentrations than did the NPNL control ewes following a 24-h fast (Figure There are two important findings of these studies. Firstly, circulating maternal insulin concentration and glucose-stimulated insulin release decrease as gestation advances. Secondly, the imposition of a two-hour hyperglycaemic clamp in pregnant ewes reduces NEFA concentrations, to concentrations not different form the pre clamp concentrations of fasted non-pregnant non-lactating ewes, though despite the reduction, insulin response to glucose remains depressed in 105 and 135 dGA ewes. These results are in agreement with the blunted insulin release during a hyperglycemic clamp treatment observed in lactating cattle, where NEFA concentrations are also elevated late in gestation, prior to a clamp ,34. GlucLipid metabolism during pregnancy has two distinct phases. The first two thirds of pregnancy are characterised by a low lipolytic activity and the majority of maternal energy appears to be derived from dietary carbohydrate. During the later part of pregnancy and into lactation, there is an increased level of lipolytic activity resulting in increased NEFA concentrations , which sin vitro secretion of insulin from pancreatic islets [These data demonstrate that the acute suppression of NEFA concentrations per se during pregnancy does not result in the restoration of a NPNL-like insulin response. This suggests that possibly other factors of pregnancy, such as the influence of lactogenic and steroid hormones, and NEFA metabolism may be acting upon the maternal pancreas to suppress the maternal insulin response during pregnancy in sheep. Placental lactogens have been documented in many mammalian species, including the sheep and human, and PL concentrations increase with advancing gestational age, reaching maximal concentrations just prior to term ,38, simic islets . Interesc islets . Studiesc islets -42. The c islets . Howeverc islets , suggestin vivo, in rat adipocyte plasma membranes incubated with GH, has resulted in a reduced adipocyte glucose uptake [Placental progesterone has also been reported to play a major role in modulating insulin release during pregnancy. In rat islet studies, progesterone counteracts lactogenic insulin stimulatory behaviour , and cire uptake , associae uptake , there ie uptake  and thate uptake ,50.in vivo, through actions in the regulation of Ca2+ influx into the β-cell [in vivo. One final possible explanation of the data reported here could be that the chronic elevation in NEFA levels, as occurs in pregnant ewes, does play a role. While the acute suppression of NEFA concentrations during pregnancy did not result in the restoration of a NPNL-like insulin response, a chronic exposure to elevated NEFAs imparts a continuous 'lipotoxic' inhibitory effect. Increased NEFA concentrations have been associated with a lipotoxic effect on the pancreas resulting in suppressed insulin release and β-cell apoptosis, following the formation of nitric oxide [Another candidate that presents itself as possibly regulating maternal insulin response is that of leptin. Leptin concentrations rise during pregnancy , and stie β-cell ,55. Howee β-cell , suggestic oxide ,19,27,28ic oxide . HoweverOne final aspect of the present studies was the attempt to use the hyperglycemic clamp parameters of MPGI, GIR and MPII ,34, to dIncreasing NEFA concentrations as gestation advances provide the maternal compartment with a double advantage. Firstly they act to an alterative fuel source for maternal metabolism. Secondly, they act to promote the development of an insulin resistant state, which is aided through the suppression of the glucose-stimulated insulin release response. This later action is still not well defined in pregnancy, but could be the result of interactions between hormones of pregnancy and the maternal pancreas, and possibly the result of chronically elevated NEFA concentrations. Through the development of insulin resistance in maternal peripheral tissues and a reduced insulin output, glucose is 'spared' and available for placental uptake and ultimately, fetal demands.NEFA Non-esterified fatty acidsNPNL Non-pregnant, non lactatingBW Body weightDM Dry matterGH Growth hormoneOPL Ovine placental lactogenAUC Area under curveCLR Glucose clearance rateMPGI Mean plasma glucose incrementGIR Glucose infusion rateMPII Mean plasma insulin increment


The aims of this randomised controlled trial are to estimate (1) the clinical effectiveness of three methods of providing mechanical support to the ankle  and usual activity, residual symptoms and need for further medical, rehabilitation or surgical treatment. Parallel economic and qualitative studies are being conducted to aid interpretation of the results and to evaluate the cost-effectiveness of the interventions.This paper highlights the design, methods and operational aspects of a clinical trial of acute injury management in the emergency department. Sprains of the lateral ligaments of the ankle joint account for between 3 and 5% of all emergency department (ED) attendances in the UK , with ap• GRADE I – the ligament is stretched but not torn and the anterior talofibular ligament is usually involved. The anterior draw test is negative.• GRADE II – the ligaments are partially torn; laxity may be present and there is moderate swelling.• GRADE III – complete rupture of the ligament resulting in joint instability. The anterior draw test is positive.The focus of this trial is Grade II and III sprains (referred to as severe sprains from this point). Severe sprains have poorer prognosis, and necessitate more intensive management than Grade I sprains (minor sprains). Minor sprains are considered to be self-limiting and require minimal treatment.® support stocking and exercise, each of which was reported as being used in most cases by over 70% of respondents [Recent systematic reviews have highlighted the lack of high quality evidence to support clinicians in managing severe sprains ,7. Therepondents . In addiPractice has been gradually changing from immobilisation of the injured joint to early mobilisation. To aid mobilisation the use of mechanical supports has been suggested. These supports vary in the amount of ankle movement they allow, but all encourage ankle flexion/extension and aim to minimise inversion/eversion that theoretically reduces the risk of further ligament injury. Movement is also proposed as a way of retraining the ankle proprioception which may reduce recurrent injury rate . Other hThe present study has two aims® ankle support and Bledsoe® boot) in comparison to Tubigrip® in1. To estimate the clinical effectiveness of three different methods of mechanical support b. The recovery of usual occupationc. Avoidance of residual symptoms including recurrent instability, lasting limitation of physical activity, and need for further medical, rehabilitation or surgical treatment.2. To measure the cost of each strategy, including treatment and subsequent health care costs.® has been chosen as the reference treatment because it is the cheapest and one of the most commonly used [® boot is lightweight and incorporates a novel design to promote ease of walking. However it is considerably more expensive, and it's clinical and cost effectiveness is yet to be proven. The below knee cast is Scotch® Cast is commonly used for casting in the NHS [® Brace.The NHS National Co-ordinating Centre for Health Technology Assessment has guided the selection of treatments. Tubigripnly used . The Ble the NHS . There iThis pragmatic randomised controlled trial is being run in 6 trusts  across the UK. The trusts are: Birmingham Heartlands and Solihull NHS Trust, North Bristol NHS Trust, Oxford Radcliffe Hospitals NHS Trust, South Warwickshire General Hospitals NHS Trust, University Hospitals Coventry and Warwickshire NHS Trust and the Worcestershire Acute Hospitals NHS Trust. Figure The target population is people attending the ED with a severe sprain of the lateral ligament complex of the ankle.People who attend with sprain of the ankle who are unable to weight bear, aged 16 years and older, and give informed consent. Weight bearing is used as a proxy for severe sprains as clinical grading is not possible in the acute phase . Flake fAge less than 16 years old, ankle or other fracture sustained in addition to the sprain . An x-ray is used to exclude fracture where this is clinically indicated and as guided by the Ottawa guidelines . People Patients are also excluded if they have a contra-indication to any of the four arms of the trial. This is most likely to occur if a patient has a DVT, high risk of DVT or other circulatory disturbance. Other contra-indications include poor skin viability preventing splinting or casting. The decision to exclude on these criteria is made by the attending clinician.A standard approach to identifying potential participants has been instituted across all participating centres. The approach was designed in consultation with the departments to dovetail with current procedures, and eradicate duplication of clinical and research data collection. The attending clinician  assesses weight-bearing status. These data are recorded on a form that includes details of the remainder of the clinical examination, including if indicated, x-ray results. At this stage, people able to weight bear are excluded.The attending clinician gives a brief explanation of the trial, an information pack, and arranges an appointment at a follow-up clinic two to three days later. Delay is an accepted practice in the application of mechanical supports, allowing for the resolution of acute swelling. It also filters out patients in whom the diagnosis of severe sprain at initial contact was incorrect, and gives participants sufficient time to consider whether they wish to participate in the trial. A physiotherapist who staffs the follow-up clinic is responsible for checking eligibility and providing a further opportunity for patients to ask questions. If appropriate, the physiotherapist recruits the patients, contacts the randomisation centre, and arranges application of the treatments. Reasons for declining to participate in the trial are recorded.Data on all ankle sprains attending the participating EDs during the recruitment period are collected using a standardised proforma. A copy of this proforma, which is entered into the medical record, is anonymised and passed to the research team. It provides descriptive data on the injury pattern, occupation, age, and sex of patients, including those who are ineligible or decline trial participation at various time points. Completed proformas are audited against ED attendance records to check whether attending clinicians have referred appropriate patients to the follow-up trial clinics. This allows clinicians who are unfamiliar with the trial to be identified and approached individually to be briefed on trial procedures.Randomisation is stratified by centre and provided via a phone-in service, which utilises a computer generated random allocation. Allocation concealment, which shields people who enter patients into a trial from knowing future allocations, is thereby ensured. The randomisation service is independently administered and quality controlled by the Birmingham Cancer Trials Unit.The mechanical supports are fitted to each individual by a trained health professional  in the follow-up clinic, to ensure comfort and correct fit. Participants are provided with standardised written and verbal instructions including when to remove the support, encouragement of normal walking with limits of tolerance, simple exercise advice, what to do in the event of experiencing difficulties with the support, and washing instructions. The protocols for duration of support, weight-bearing status, and activity have been determined by the manufacturers recommendations and the results of a national survey of practice completed in the planning phase of the trial . TreatmeAll other treatments are standardised and include the provision of crutches, advice to elevate and pain relieving medications if needed. Withdrawal of the latter treatments would be inappropriate as they constitute normal and accepted care. Physiotherapy is not provided as part of the trial treatment protocol, and is not part of routine care provided in the participating centres . If the Clinical status is measured at baseline, 4 weeks, 12 weeks and 9 months. The baseline assessment also includes date of birth, sex, body mass index, ethnicity, an assessment of pre-injury abilities including usual levels of mobility, engagement in sporting activity, usual occupation and employment (including hours worked and type of work undertaken) and completion of the baseline version of the outcome questionnaires.The selection of outcome measures is based on a systematic review completed during the preparatory stage , and areThe mechanical supports are likely to have maximal impact and benefit during the first three months of recovery, and this time period is defined as the primary time point. We anticipate that some participants will still be wearing the mechanical support at 4 weeks. The natural time course of recovery of ankle sprains is for functional limitations to stabilise between 3 and 9 months and it is expected that the difference between treatments will narrow in the longer term as the majority of people will recover ,6. PartiFollow-up data are collected by postal questionnaire. We have implemented an intensive approach to follow-up. Participants are mailed a follow-up questionnaire, and if a response is not received within one week, they are telephoned to ensure they have received the questionnaire. A second questionnaire is mailed if necessary. If there is still no response, the participant is contacted one week later, and a core set of outcomes are collected over the telephone. All follow-up data are collected and analysed by individuals who are independent of the recruitment, randomisation, baseline assessment and are masked from the treatment provision. This level of masking will be maintained until final analysis of the data has been completed. The only exception to this rule will be if the data monitoring committee require unmasked data, and in this circumstance, only the independent Chairperson will be aware of assignments.Trial procedures are audited at regular intervals to ensure compliance with the protocol. Maintaining trial profile and competence in trial procedures is challenging in the ED, because of a high turn-over of staff and a diversity in the staff groups dealing with injuries in different hospitals. To combat these difficulties, training is provided on cycles that coincide with staff rotation, as well as various other time points (e.g. in-service training).The analysis will be conducted as intention to treat. An analysis of all people who completed the trial will be undertaken, and in addition, a sensitivity analysis will be undertaken to assess the range of potential biases that could result from loss to follow up or withdrawal. Numerical and graphical summaries of all the data will be compiled, including a detailed description of missing data at the clinic visit, questionnaire and individual level.®, so each of the three other treatments will first be compared with it. Any treatments found to be more effective than Tubigrip® will be compared with each other. Regression modelling will allow an assessment of factors that might indicate the appropriate choice of treatment. As these analyses are pre-specified, issues of multiple comparisons are minimal.Logistic and log-linear multinomial regression models will be used to provide estimates of the recovery rates and the prevalence of residual symptoms, with confidence intervals. As there are multiple centres and repeat assessment, random effect  models will be used to investigate the components of variation. The common current and cheapest therapy is TubigripThe sensitivity of the above analyses to missing data at various levels will be assessed and quantified using modern statistical methods for incomplete multivariate data .Severe sprains may have a range of direct cost consequences across primary and secondary health care, they may also have cost consequences for patients themselves in terms of their personal expenditure and return to work. The costing study will seek to adopt a broader societal perspective, including patient costs, when estimating differences in the cost of resources used in the four arms of the trial. The economic analysis will compare resource use (costs) with any measurable changes in health outcomes (benefits). The health outcomes of the four technologies will be measured in terms of changes in specific disease parameters and validated measures of health-related quality of life (HRQL). Any uncertainties in the cost and outcomes data will be incorporated into a sensitivity analysis.The cost of each mechanical support protocol will be determined through observation and will include staff time, overheads, equipment and transport; plus follow-up visits to hospital, GP surgeries, physiotherapists or others. The consequences for patients in terms of GP and hospital visits, including travel expenses and time off work, and expenditure on aids or private practitioner input are obtained from structured resource use questions added to patient follow-up questionnaires. Further treatments recorded include pain relieving medications, anti-inflammatory and other topical agents, bandages, supports or footwear. Patients are also asked to distinguish whether these are NHS-funded or private treatment paid for by the individual or private provider. Patient self-reported information on service use has been shown to be accurate in terms of intensity of use of different services . HospitaThe appropriate technique of economic evaluation will depend on the results of the study . The simAlthough there was a paucity of published data available to inform the sample size estimate at the outset of the trial, the preliminary phase of the study has enabled us to make a more accurate estimation of the sample size required. The presented estimate is based on a standard sample size calculation for a two-sample t-test with equal variances and a significance level of 0.05, using the variance estimated from an ANOVA of the 4-week data (n = 100). The minimal clinically important difference is set at 10 percent, which represents a small to moderate effect size. A target of 600–650 participants will allow us to detect clinically important outcomes at 4 and 12 weeks. We are powered >90% to detect differences of 10 percent in the primary outcomes, and have sufficient power to detect differences in a range of secondary outcomes at 80% power. To account for the possibility of loss to follow-up, the estimate includes an inflation of 20%. However, the trial should be able to report whether any of the treatments have a sustained negative effect on outcome at 9 months. We are taking a pragmatic approach to estimating the sample size, and the Data Monitoring Committee are reviewing assumptions underlying the calculation at 6-monthly intervals.A purposive sample of randomised patients will be interviewed to obtain qualitative data on the patients' experiences of the various treatments in a semi-structured interview. Patients will also be asked for their views on their willingness to pay a deposit for the boots/splints to encourage their return. Up to 20 patients will be interviewed using a semi-structured format. The data will be analysed using thematic content analysis.We have described the protocol and conduct of a large scale UK randomised controlled trial of mechanical supports for the management of acute severe ankle sprains. There has been a paucity of good quality research conducted to date, which may be partly explained by the challenges implicit to studying acute events within EDs ,25. ThesThe author(s) declare that they have no competing interests.SEL wrote the original protocol, secured funding, act as co-principal investigator and lead in the writing of this manuscript. MWC wrote the original protocol, secured funding and act as co-principal investigator. JRD and SW contributed to the development of the protocol and securing funding. JLH and AS contributed to the development of the protocol and securing funding and wrote the statistical and economic aspects of the protocol respectively. RAN is responsible for the day to day co-ordination of the trial, has contributed to refining the protocol and outcome measure package. JLM revised the protocol and the sample size calculations. MC refined the economics section of the protocol and is providing economic input. EJW is responsible for the data management and trial support. All authors have contributed to and approved the final manuscript.Lamb SE, Cooke MW, Nakash RA, Withers EJ, Hutton JL, Marsh JL, Szczepura A, Wilson S, Clark M, Dale JR, MacNamara A, Kendall J, Skinner D, Sakr M, Kelly C, O'Byrne G, Morrell R, Dunn M, Bateman S, Kempson S, Hooker F, Parker N, Noakes E, Vaux N, Doughty G, Nichols V, Ritchie C, Sabine E.Professor Bill Gillespie, Professor Sallie Lamb, Dr Matthew Cooke, Professor Jeremy Dale, Professor Jane Hutton, Dr Jen Marsh, Professor Ala Szczepura, Dr Sue Wilson, Rachel Nakash, Vicki Staples.Dr Janet Dunn, Professor Damian Griffin, Pat Overton-Brown.The pre-publication history for this paper can be accessed here:


There is a small, but growing body of literature highlighting inequities in GP practice prescribing rates for many drug therapies. The aim of this paper is to further explore the equity of prescribing for five major CHD drug groups and to explain the amount of variation in GP practice prescribing rates that can be explained by a range of healthcare needs indicators (HCNIs).The study involved a cross-sectional secondary analysis in four primary care trusts (PCTs 1–4) in the North West of England, including 132 GP practices. Prescribing rates (average daily quantities per registered patient aged over 35 years) and HCNIs were developed for all GP practices. Analysis was undertaken using multiple linear regression.Between 22–25% of the variation in prescribing rates for statins, beta-blockers and bendrofluazide was explained in the multiple regression models. Slightly more variation was explained for ACE inhibitors (31.6%) and considerably more for aspirin (51.2%). Prescribing rates were positively associated with CHD hospital diagnoses and procedures for all drug groups other than ACE inhibitors. The proportion of patients aged 55–74 years was positively related to all prescribing rates other than aspirin, where they were positively related to the proportion of patients aged >75 years. However, prescribing rates for statins and ACE inhibitors were negatively associated with the proportion of patients aged >75 years in addition to the proportion of patients from minority ethnic groups. Prescribing rates for aspirin, bendrofluazide and all CHD drugs combined were negatively associated with deprivation.Although around 25–50% of the variation in prescribing rates was explained by HCNIs, this varied markedly between PCTs and drug groups. Prescribing rates were generally characterised by both positive and negative associations with HCNIs, suggesting possible inequities in prescribing rates on the basis of ethnicity, deprivation and the proportion of patients aged over 75 years . The aim of this paper is to provide data on the equity of general medical practitioner (GP) prescribing rates for coronary heart disease (CHD) drugs. Since CHD represents a major cause of premature mortality in the Western world, it is vital that those populations with the highest need for CHD drugs actually receive them. Whilst there is a large literature on inequities in the provision of a number of other health care services and treatments, the equity of GP practice prescribing has received little attention. Therefore, this study was an attempt initiate the development of an evidence-base and to provide data on the equity of GP practice prescribing rates.access to health care for people in equal need; equal treatment for people in equal need; and equal outcomes for people in equal need [There are large literatures around how to define, operationalise and measure equity in relation to health care services -3, althoual need . Whilst treatment) for people in equal need. Using the example of the current study, an analysis of equity would assess the differences in prescribing rates provided to the population of one GP practice compared to another GP practice, weighted to take account of the levels of need for CHD drugs in their patient populations. Therefore, it would be equitable to have higher prescribing rates for populations with higher levels of health care need and lower prescribing rates for populations with lower levels of health care need. However, it would be inequitable to have higher prescribing rates for populations with lower levels of health care need and lower prescribing rates for populations with higher levels of health care need. The identification of populations where prescribing was deemed inequitable could then be targeted for further resources aimed at redressing the balance between prescribing rates and health care need.The current paper is focussed around the equal prescribing . Essentially, PCTs are organisations whose main responsibilities are around developing, commissioning and providing services, which are targeted to the needs of local people, and ultimately to improve the health  of local people ,26. PCTsThis study was undertaken in 4 PCTs in England , which included 132 GP practices . In terms of patient populations, we excluded patients aged under 35 years, since prevalence of CHD is particularly low in this age group. In total, there were 353,897 registered patients aged over 35 year across all 4 PCTs.In order to analyse the equity of prescribing for CHD drugs, we firstly needed to gather data on, and then develop rates for both GP practice prescribing and health care need  in this paper). The data sources and methods used to develop prescribing rates and HCNIs have been outlined in previous papers by the authors ,29,30, aWhen an NHS prescription is dispensed in primary care, the prescription form (FP10) is sent to the Prescription Pricing Authority (PPA) for processing. The PPA collates these data and provides them to GP practices and PCTs in the form of Prescribing Analysis and Cost (PACT) data. PACT data are available for all GP practices in England, and allow detailed interrogation in terms of drugs prescribed along with their dosages, pack sizes and formulations. For example, for a specific time period, we can collect data on which statins were prescribed by a GP practice in addition to the dosages and pack sizes. This allows for a complex and timely analysis of PACT data. Useful critiques of PACT data can be found elsewhere ,32.PACT data were obtained for all GP practices in the 4 PCTs for the 12-month period October 1999 to September 2000. These data were collected for statins, ACE-inhibitors, beta-blockers, aspirin, and bendrofluazide (a full list of drugs obtained are listed in Appendix A). These drug groups were chosen because they represent major drug groups recommended for the prevention (primary and secondary) of coronary heart disease (CHD) in the United Kingdom (UK) . Using p and ADQs  for all drugs for which they have been developed. Within this study, total ADQs were used as the unit of analysis since they represent prescribing practices in the UK, as opposed to DDDs which represent prescribing practices internationally.The numerator in all prescribing rates was based on a measure of prescription volume, as opposed to prescription cost. The validity of using the number of prescription items or total cost as a measure of prescribing volume has been called into question ,39 sinceThe denominator was the total registered (and resident) patient population aged over 35 years. This age group was chosen since the prevalence of CHD is particularly low in people aged less than 35 years .In total, 24 HCNIs were developed for each GP practice in this study, and all of these were entered into multiple regression models . These HCNIs reflect patient demographics, ethnicity, socio-economic status, long term limiting illness, CHD mortality and CHD morbidity. However, the only HCNIs discussed here are those included in the final regression models  the health care need in GP practice populations. In terms of coronary heart disease (CHD), there are a number of identifiable risk-factors, all of which increase the risk of CHD morbidity and/or mortality. However, readily available data are not available for a number of these risk-factors, and therefore could not be explored within this study. Nevertheless, data were available on the following risk-factors: age, gender ethnicity, socio-economic status. Evidence on the importance of these risk-factors for assessing health care need is presented below.Rates of CHD related morbidity and mortality increase dramatically with age ,46. For Rates of CHD mortality and morbidity in the UK are generally seen to be higher for people from South-Asian groups, than from white-English people, and much lower for people born in the Caribbean. It has been suggested that the mortality rate for South Asians is 50 per cent higher than for the general population . In the There is an extensive literature on socio-economic inequalities in CHD mortality and morbidity -57. WhilOverall, the age, ethnicity and socio-economic status are all important factors in shaping the epidemiology of CHD in the UK, and as such, are important variables for use in developing the HCNIs in this study. The actual development of HCNIs relating to these risk factors are outlined below, in addition to additional HCNIs related more directly to CHD morbidity and mortality.Some HCNIs were collected directly from GP practice lists (proportion of patients aged over 75 years and proportion of patients aged 55–74 years), some have been calculated for GP practices (Low Income Scheme Index (LISI) score) , and othDemographic HCNIs were developed directly from GP practice list data, and these relate to the proportion of patients aged 55–74 years, and the proportion aged over 75 years. Both of these demographic groups are indicators of health care need for CHD drugs . The LowData were also obtained from hospital episode statistics (HES) on specific hospital procedures (coronary artery bypass graft (CABG), percutaneous transluminal angioplasty (PTCA), and coronary angiogram) and diagnoses (primary diagnosis of CHD at discharge). Although HES relate to the supply, as opposed to need for health care services , it was As outlined in Appendix B, a number of other variables relating to CHD mortality and morbidity were entered into the regression models, although these were not statistically significant. Indeed, as outlined in a previous paper by the authors , bivariaMultiple linear regression modelling was undertaken for each drug group in each PCT in addition to the combined dataset. The dependent variable in each model was the respective prescribing rate and the independent variables were all 24 HCNIs developed in the study. The forward-stepwise approach was taken but not slavishly adhered to since considerations of model coherence and plausibility were taken into account when seeking the most parsimonious models. The final regression models only contained the HCNIs which had statistically significant (p < 0.05) independent associations with the dependent variable, and each model was checked for collinearity and normality of residuals. Overall, for each multiple regression model, all 24 HCNIs were entered as independent variables, and the final model included only those variables that were statistically significant and added to the fit of the model.Descriptive findings will be presented first, in order to contextualise the findings from the multiple regression analyses.Table PCT1 had the highest median prescribing rates for all drugs except aspirin, where PCT4 had the highest median rate. PCT3 had the lowest median prescribing rates for aspirin, beta-blockers and ACE inhibitors, and PCT4 had the lowest median prescribing rates for statins, bendrofluazide, and all study CHD drugs combined. The difference between the median prescribing rates for PCT1 and PCT4 for bendrofluazide, statins and all CHD drugs was around 4, 5, and 21 ADQs per patient over 35 years. Therefore, an average GP practice in PCT1 with 3000 registered patients over 35 years, prescribed 12000 more ADQs for bendrofluazide, 15000 more ADQs for statins, and 63000 more ADQs for all study CHD drugs. In addition, a similar GP practice in PCT1 also prescribed almost 30000 more ADQs for ACE inhibitors than a comparably sized GP practice in PCT3.An initial overview of the health care needs of the PCTs suggests that PCT4 had the highest levels of CHD related health care needs within the study, whereas PCT1 had the lowest health care needs. PCT4 was the most deprived of all PCTs, had the highest proportions of patients aged over 75 years, and the highest median score for standardised mortality rates (SMR) for CHD. In contrast, PCT1 may be seen as the 'least needy' of all PCTs on the basis of the HCNIs developed in this study. PCT1 was the least deprived, had the lowest proportions of South Asian groups, had the lowest median SMR and the lowest median rate of CHD hospital procedures.These overall descriptive findings are indicative of inequitable prescribing rates, whereby PCT1 had high prescribing rates and low comparative health care needs, whereas PCT4 had low prescribing rates and high comparative health care needs. However, these are purely descriptive and do not take into account the multiple risk factors for CHD or the interactions between HCNIs. Therefore, findings from the multiple regression analyses are presented below.2 * 100), Table 2 of the model.Table 2 values in general, and no model was derived for ACE inhibitors. Given the reduced explanatory power of the regression models in PCT1, when regression modelling was undertaken for the combined dataset, the R2 values were much lower than those for PCT2, and generally lower than those for both PCT3 and PCT4.Regression models generally explained much more variation in prescribing rates in PCT2 than any other PCT. For example, in PCT2, modelling explained around 55 per cent of the variation in prescribing rates for ACE inhibitors and bendrofluazide, almost 60 per cent for statins and almost 90 per cent of variation in aspirin prescribing rates. Multiple regression models explained around 25 to 55 per cent of the variation in prescribing rates in the combined dataset, around 25 to 60 per cent in PCT3, and around 40 to 55 per cent in PCT4. PCT1 had the lowest RIn general, regression modelling explained more variation in prescribing rates for aspirin than for the other drug groups. Not including PCT1, regression modelling explained between 50 and 90 per cent of the variation in aspirin prescribing rates. Comparative percentages were between 30 and 60 per cent for statins, 30 and 45 per cent for beta-blockers, and 25 and 55 per cent for ACE inhibitors and bendrofluazide.Table The models for statins, aspirin and ACE inhibitors also included the proportion of patients aged over 75 years although this indicator had a negative association with prescribing rates for statins and ACE inhibitors, and a positive association with prescribing rates for aspirin. This suggests that GP practices with higher proportions of patients aged over 75 years have lower prescribing rates for statins and ACE inhibitors, and higher prescribing rates for aspirin.The CHD HES rate varied considerably between models, in terms of the amount of variation explained. For example, it explained 33 per cent of the variation in aspirin prescribing rates, 20 per cent of the variation in prescribing rates for all CHD drugs and beta-blockers, 15 per cent of the variation in statin prescribing rates, and 7 per cent of the variation in prescribing rates for bendrofluazide.The LISI score explained 23 per cent of the variation in prescribing rates for all CHD drugs, 13 for bendrofluazide and 10 per cent for aspirin. In addition, the proportion of patients aged between 55 and 74 years explained between 2.5 and 7 per cent of the variation for statins, ACE inhibitors, beta-blockers, bendrofluazide, and all CHD drugs.The rate of CHD hospital procedures (CHD HES) explained some of the variation in prescribing rates and therefore, prescribing rates in these instances were positively related to health care need (i.e. equitable). These findings concur with other studies whereby positive relationships were found between rates of PTCAs and both aspirin prescribing  and statThe proportion of patients aged 55–74 years was generally positively related to prescribing rates, suggesting that prescribing rates are related to health care need, since people in this age group have a higher prevalence of CHD . A numbeWhilst the previous finding is not unexpected given the higher CHD prevalence in this age group, the negative relationship between the proportion of patients aged over 75 years and some prescribing rates should be of great interest. The proportion of patients aged over 75 years had a negative relationship with statins and ACE inhibitors and a positive association with aspirin. A number of studies found that older patients were much less likely than younger patients to undergo a CABG or a PTCA,72 or to2 values reveal a large amount of unexplained variation in prescribing rates. Indeed, even with the models for aspirin and all CHD drugs, there is still around 50 per cent of unexplained variation in prescribing rates.Overall, this study has found that prescribing rates may be explained (to differing degrees between primary care trusts (PCTs) and between drug groups) by a mixture of health care need indicators (HCNIs). Whilst prescribing rates were generally positively related to the rates of hospital procedures and diagnoses, they were also negatively associated with proxies of deprivation and ethnicity and with the proportion of patients aged over 75 years. However, the relatively low RThis study cannot be used to infer inequitable prescribing by GPs, since the lower prescribing rates in areas with high proportions of South Asian and deprived groups may be due to lower utilisation of primary health care services due to social, economic or cultural barriers ,75. TherWhilst the clinical and epidemiological data on these CHD drugs has allowed for the development of evidence-based guidelines and evidence-based prescribing, this paper suggests that in practice, actual prescribing rates may not be related to health care need. Further research needs to concentrate on verifying or falsifying these claims on a more micro-level analysis  and on exploring the reasons why such a relationship exists . In addition, more work is required to understand the differences between PCTs in terms of the explanatory power of the regression models (i.e. much more of the variation in prescribing rates were explained in PCT2 than any other PCT). Such a strategy may enable educational tools to be developed which would facilitate more evidence-based prescribing, but may also identify particular patient groups who do not present symptoms of CHD (ie unmet need) and therefore may require educational outreach or targeted screening in order to increase their consultations and ultimately prescribing to these groups. Although we have focussed on drugs used in the prevention of CHD, a similar approach may be taken across different therapeutic groups of drugs, in different health care settings and in different countries in order to generate more rounded, grounded and extensive evidence on the equity of prescribing in general.• Aspirin (75 mg)1 (2.5 mg)• Bendrofluazide• Statins 2 • ACE inhibitors3 • Beta-blockers1 In some countries, this may also be known as (among other names) Neo-NaClex, Bendroflumethiazide, Aprinox, Berkozide, Naturetin, Pluryl, Polidiuril, Salural, Urizde.2 The 5 ACE inhibitors represent the majority of prescribing for all ACE inhibitors3 Atenolol represents the majority of prescribing of all beta-blockers4 Co-tenidone is a combination product containing both a beta-blocker (atenolol) and a diuretic .• Proportion of patients aged between 55 and 74 years• Proportion of patents aged over 75 years• Proportion households with no car• Proportion males who are economically inactive• Townsend Score• Proportion of households receiving council tax benefits• Proportion unemployment• Index of Multiple Deprivation• Income Deprivation Index• Low Income Scheme Index (LISI) score• Standardise mortality rate (SMR) for CHD under 75 years• 6-year crude rate of coronary artery bypass grafts (CABGs) per 1000 patients• 6-year crude rate of percutanious transluminal angioplasty (PTCAs) per 1000 patients• 6-year crude rate of coronary angiograms per 1000 patients• 6-year crude rate of CHD hospital procedures (CABGs + PTCAs + angiograms) per 1000 patients• 6-year crude rate of CHD hospital diagnoses per 1000 patients• 6-year crude rate of CHD prevalence (diagnoses + procedures) per 1000 patients• Regionally specific prevalence, age and sex standardised prescribing units per patient over 35 years (PASS-PUs)• Proportion of population defining themselves as 'non-white'• Proportion of population defining themselves as 'South Asian'• Proportion of population over 30 with a limiting long-term illness (LLI)• Health Deprivation Index• Proportion of households with more than 2 cars• Access Deprivation IndexThe author(s) declare that they have no competing interests.PW was awarded funding for this study, was involved in the conception, design and managed the day to day running of the study, undertook all data collection and analysis, and wrote the paper. PN and ASL were involved in the conception and design of the study, made active contributions in project meetings, were involved in an advisory capacity in all aspects of the project and advised on drafts of the paper. PW is the guarantor.


Diagnosis of HIV infection is recently occurring with increasing frequency in middle-aged and in older individuals. As HAART became available, a minimal beneficial effect on immunological outcome in older in respect of younger subjects has been reported. In fact, both the intensity and the rapidity of the immunological response appeared to be reduced in elderly subjects. On the contrary, only few reports have indicated a similar immunological outcome both in older and younger HIV-positive subjects. Interestingly, older age did not seem to significantly affect the long-term virological outcome of HAART treated subjects.To characterise epidemiological and clinical features of older HIV+ subjects, a prospective case-control study was performed: 120 subjects ≥ 50 and 476 between 20 and 35 years were initially compared. Subsequently, to better define the impact of HAART on their viro-immunological response, 81 older were compared with 162 younger subjects.At baseline cases presented significantly lower TCD4+ cell number and were more frequently affected by comorbid conditions. Under HAART a statistically significant increase in TCD4+ cell number was observed in cases and controls. At multivariate analysis, there was no statistically significant difference between cases and controls regarding viro-immunological response.Although older subjects present a more severe HIV infection, they can achieve, under HAART, the same viro-immunological success as the younger individuals. Early in the epidemic, Human Immunodeficiency Virus (HIV) infection primarily affected young adults; later on, it occurred with increasing frequency in middle-aged and in older individuals, too. In fact, a rate of 12.3% of AIDS patients aged 50 years or older, with subjects ≥ 60 years equalling 3.2%, has been recently reported by the Centres for Diseases Control and Prevention (CDC) [Most of the known epidemiological features about older HIV-infected subjects, have been defined before the introduction of Highly Active Antiretroviral Therapy (HAART), as the standard therapy for HIV-positive subjects, in 1996. A low level of T CD4+ cells at HIV diagnosis ,3, a rapAs HAART became available, a less beneficial effect on immunological outcome in older in respect of younger subjects has been reported -17. In fTwo were the aims of this study:1. To characterise the epidemiological and clinical features of older subjects at the time of HIV diagnosis in the HAART era.2. To better define the impact of HAART on virological and immunological response in a cohort of older HIV-positive patients when confounding variables such as adherence to therapy, side effects and non HIV-related comorbidities were evaluated.The Catholic University teaching hospital is a 1,900-bed tertiary care centre located in Rome, Italy, with approximately 67,000 patient admissions each year. There is a 60-bed unit for the admission of HIV-infected subjects and a day-hospital for out-patient care.Prospective case-control study.All patients presenting the first HIV positive test from January 1997 to June 2003, admitted to our in- and out-patient care, were considered.For the first aim of the study, subjects aged ≥ 50 years (older) and between 20 and 35 years (younger) were identified.For the second aim of the study, older and younger patients who were given HAART regularly and with a follow-up of at least six months, were included as cases and controls, respectively (ratio 1:2). The control group was matched by sex, year of HIV diagnosis, presence of AIDS defining conditions and type of HAART regimen (i.e. NNRTI containing vs. PI containing regimens). A patient (either case or control) was considered as regularly HAART-treated if he/she was under antiretroviral therapy with a combination of two nucleoside reverse transcriptase inhibitors (NRTI) plus either a non nucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor (PI), as single drug or with the addiction of a low dose of ritonavir (i.e. 100 mg bid), for at least three months.Pneumocystis carinii pneumonia (PCP) and toxoplasmosis, antiretroviral therapy  and other medications , time of follow-up, time from AIDS diagnosis to death, cause of death.Patients' records were reviewed by one of us, using a standardised data collection form with predefined criteria. For each subject enrolled in the study, the following data were obtained at inclusion after patient's informed consent: age, gender, HIV-risk behaviour, date of the first available HIV test, reason for HIV testing, CDC stage of HIV infection , HIV-rel3), CD8+ T cells (/mm3) and plasma HIV RNA viral load (copies/ml) with a limit of detection below 50 copies/ml. The time of the beginning of HAART was considered as time zero in the analysis. All subjects underwent periodical clinical evaluations including determination of CD4+ T cells and HIV viral load every six months.Data collected from the laboratory records included: numbers of circulating CD4+ T cells (/mm3).Adherence was defined through a self-reported evaluation by the patient and registered as percentage by the physician. An evaluation higher than 80% was classified as "adherent", whereas lower than 80% was considered as "non-adherent". The following adverse effects were considered: anaemia (haemoglobin <9.0 gr/dl), digestive intolerance, dyslipidemia (serum cholesterol >240 mg/dl and/or triglycerides >200 mg/dl), hepatitis, hyperglycaemia (glucose >150 mg/dl), lipodystrophy, pancreatitis, peripheral neuropathy, rash, renal colic and thrombocytopenia  defined as a HIV viral load undetectable (<50 copies/ml) at the end of the follow-up; 3. Viro-immunological success (VIS) defined as either undetectable HIV viral load and T lymphocytes CD4+ cell count >200/mm3 at the end of the follow-up.Three outcomes were considered for the study: 1. Immunological success (IS): defined as T lymphocytes CD4+ cell count >200/mmIn order to evaluate the significance of non HIV-related comorbid conditions, we used a modified version of the Charlson comorbidity index which excludes AIDS from the diagnosis . Cardiov2 test or, for small numbers, Fisher's exact test. Stepwise logistic regression models were used for each factor to adjust for the effects of confounding variables. Two tailed tests of significance at the p < 0.05 level were used to determine statistical significance. Statistical analysis was performed using the software program Intercooled Stata 6.0 .Quantitative variables were tested for normal distribution and compared by means of Student's two tailed t-test. Differences in group proportions were assessed by use of the χThe rate of HIV diagnosis in patients aged ≥ 50 years admitted to our in- and out-patient care remained constant (about 10%) over the study-period.From a total of 1265 subjects with new HIV-positive test, 129  subjects aged ≥ 50 years were identified. Nine of them were not further considered because of missing data. One hundred twenty older subjects were evaluated. Data were available for 476 younger subjects.Among older subjects there were more males, less intravenous drug abusers, more advanced HIV disease, more frequent C stage of HIV infection and higher mortality, Table .The reasons for the first HIV test were comparable both for older and younger subjects. In particular, 30 (25%) older subjects were asymptomatic. For those who were symptomatic, the more frequently observed symptoms were: fever (25%), haematological dysfunctions (17%), weight loss (14%), neurological (8%), gastrointestinal (6%) or hepatic (7%) disorders.Regarding AIDS-defining conditions, a significantly higher frequency of PCP, HIV-encephalopathy, Kaposi's sarcoma and cryptococcosis was observed in older HIV-infected patients than in younger.Thirty nine out of 120 HIV-positive older subjects were not further considered: 29 because the duration of follow-up was below six months and ten because the case-control matching was not possible. A final number of 81 older patients under regular HAART and with a follow-up of at least six months (cases) were thus compared with 162 younger subjects (controls).3 vs. 178 ± 189 cell/mm3; p < 0.01), such as the mean CD4 percentage . The mean of CD8+ T cells at baseline was similar in cases and controls (757 ± 380 cell/mm3 vs. 818 ± 362) (p = ns). Mean of HIV viral load log was similar in the two groups . No statistical significant difference was observed for haematological parameters, serological markers of previous viral hepatitis (type B or C) and syphilis. Laboratory tests only indicated a significant increase of glycaemia in older patients .Cases had a median age of 56.9 ± 5.2 years and controls of 31.3 ± 3.2 years. As a result of the matching, 73% of subjects in both groups were males and 49% were in CDC stage C of HIV infection. HIV infection risk factors were similar in the two groups, but eighteen (22%) cases referred an unknown risk factor compared to 26 (16%) controls (p = 0.1). The mean of CD4+ T cells was significantly lower in cases compared to controls , ranging from 6 to 78 months.No differences were observed between cases and controls in the number of hospitalisations .Seven (9%) and six (4%) patients died in the case and control groups, respectively (p = ns). All causes of death in both groups were HIV-related conditions with the exception of one case who died for an acute mesenteric vascular episode.Cases had more comorbid conditions than controls . One third of the cases suffered from cardiovascular diseases, a rare condition among controls . Gastrointestinal disorders  and diabetes  were also significantly more frequent among cases than controls. No statistically significant differences were found for cancer, pulmonary, renal or endocrine diseases, chronic hepatitis and coagulation disorders. The total comorbid points were 37 for cases and 20 for controls. Cases had also a higher mean Charlson index in respect to controls .No statistically significant difference between cases and controls was observed in type, number and duration of HAART regimens. The first line antiretroviral therapy included PI in more than 80% of cases and controls Table . The patComparing the mean values obtained at baseline in both cases and controls with those obtained at the end of the follow-up, a statistically significant increase in T CD4+ cell number was observed. In particular, T CD4+ cell count increased in 48 months from 107 ± 109 to 476 ± 258 for cases (p = 0.04), and from 178 ± 189 to 584 ± 252 for controls (p < 0.01). There was always a progressive linear enhancement in both groups at any individual time of the follow-up. The lower values observed in cases in respect to controls at baseline, persisted until the end of the follow-up , enhancement of CD4+ T cell count in the first six months (p < 0.01), HIV viremia at 30 months (p < 0.01).According to the above mentioned outcome definition, IS was observed in 59 (73%) of the cases and 128 (79%) of the controls (p = ns). The following variables were significantly associated with IS at univariate analysis: CDC stage A (p = 0.01), low Charlson index (p = 0.01), CD4+ T cell count at the beginning and at 6,12,18,24,30,36 months of HAART (p < 0.01), months to achieve CD4+ T cell >200/mmA statistically significant reduction of HIV viral load was observed in both cases and controls when comparing baseline with the end of the follow-up values. In particular, means of HIV-RNA viral load log decreased in 48 months from 4.74 ± 0.57 to 2.01 ± 0.4 copies/ml in cases (p < 0.01) and from 4.69 ± 0.76 to 1.6 ± 0.2 copies/ml in controls (p < 0.01).HIV viral load reduced under antiretroviral therapy in both groups in a comparable manner. In fact, there was no statistically significant difference between cases and controls at any individual time of the follow-up Figure .According to the above mentioned outcome definition, VS was observed in 66 (82%) of cases and 121 (75%) of controls (p = ns). The following variables were significantly associated with VS at univariate analysis: CDC stage A (p = 0.02), high adherence to HAART (p = 0.01), dyslipidemia as adverse effect of HAART (p = 0.01), HIV viral load at 6 (p < 0.01) and 24 months (p = 0.02).According to our definition of the outcome, VIS was observed in 53 (66%) of cases and 100 (62%) of controls (p = ns). The following variables were significantly associated with VIS at univariate analysis: CDC stage A (p < 0.01), high adherence to HAART (p = 0.02), dyslipidemia as adverse effect of HAART (p < 0.01), CD4+ T cell number at baseline and at 6,12,18,24,30,36 months of HAART (p < 0.01), enhancement of CD4+ T cellcount in the first six months (p < 0.01), HIV viral load at 6 (p = 0.04) and 30 months (p = 0.01).No statistical difference in immuno-virological response was found between cases and controls when, as cases, only a subgroup of patients aged > 60 years was considered (data not shown).In a multivariate logistic regression model, after adjustment for sex and Charlson index, there was no statistically significant difference between cases and controls for IS, VS and VIS (p = ns). Similar results were obtained when CDC stage A and CD4+ T cell count at HIV diagnosis were added to the model.Another model including sex, Charlson index, CDC stage A, CD4+ T cell count at the beginning of HAART, adherence and dyslipidemia as adverse effect of HAART did not show any statistically significant difference between cases and controls for IS, VS and VIS.3, HIV viral load at six months did not modify the results.Further adjustment for sex, Charlson index, CDC stage A, adherence, dyslipidemia as adverse effect of HAART, CD4+ T cell count at beginning of HAART and after six months of therapy, enhancement of CD4+ T cell count in the first six months, months to achieve CD4 + T cell count >200/mmThis study, conducted in an Italian population, shows that in the HAART era, the percentage of older patients out of the new HIV infections is approximately 10%, slightly higher than previously reported . The impThe first part of our study confirms previous pre-HAART era observations, about more severe HIV infection in older subjects -10. In fRegarding HIV-risk behaviour, our data indicate the heterosexual route as the most frequent in older patients, followed by no risk reported, homosexual contacts, intravenous drug abuse. In particular, our data confirm recent reports ,27 indicTo date, no studies have indicated that older AIDS patients should be treated differently for HIV disease. Clinical drug trials often exclude older patients because of multiple medical problems, comorbidities or co-existing non HIV-related medication regimens . The firVery little literature and study has been devoted specifically to the role of protease inhibitors and aggressive antiretroviral triple therapy in elderly patients. Few data have been published concerning the specific response of older patients to new HIV antiretroviral treatments, and currently there are no guidelines for specific antiretroviral treatment modalities for patients>50 years of age. However, a poor immunological response under HAART was observed in small groups of patients ,16 possiThis six year prospective matched case control study not only confirms this observation but indicates that older patients under HAART experienced a successful immunological response comparable to younger subjects also in a long term follow-up, in line with preliminary reports -34. In fMeticulous adherence to HAART is necessary to achieve an optimal clinical and virological response. A variety of factors may predict medication adherence among HIV-infected adults. Although older age is usually associated with higher rates of antiretroviral adherence, older participants who were cognitively impaired could have difficulty in adequately adhering to their medication regimen -37. Our Early work also demonstrated a positive association between age and the plasma HIV-1-RNA copy number at the time of identification of HIV-1 serostatus among recently diagnosed older HIV-1 positive individuals . HoweverNo differences were observed in virological response as indicated by viral load reduction in older patients with respect to younger. This result is in line with previous reports ,18 incluIn our study both the frequency and the distribution of adverse effects of HAART were similar between the two groups, with the exception of anaemia and hyperglycaemia which were more frequent in older patients. Hyperglycaemia can be explained because diabetes is a frequent comorbid condition among older. Older subjects were more susceptible than younger to adverse effects in the first line therapy; consequently they had to change therapy more frequently.Univariate analysis showed an association of low Charlson index with immunological success. However, considering that the Charlson index was higher in older patients, none of the models of multivariate analysis indicated an association between comorbidity and virological and/or immunological response. Nonetheless previous studies have suggested a more rapid course and a decreased survival in older HIV-positive patients because of the presence of comorbidities , our datDyslipidemia and lipodystrophy in association with gastrointestinal disorders were the more frequent adverse reactions. In view of these results, of the recent reports of endothelial vascular damage, premature atherosclerosis and coronary events in younger HAART-treated patients -45, an iIn conclusion, we strongly underline the need for an early diagnosis of HIV infection in older subjects through implementation of educational campaigns specifically targeted to this population. In relation to older people, physicians should consider more often the possibility of HIV diagnosis, through more screening and counselling. An early diagnosis is mandatory because, although older subjects present with more severe HIV infection, the use of HAART allow them to achieve the same viro-immunological response as younger individuals. At present, there is an increasing number of older subjects living with HIV either because of new HIV diagnosis in older population or because who have previously acquired HIV infection now become older due to HAART improved survival. It is reasonable to predict that the average age of chronically treated HIV-positive patients will progressively increase in the forthcoming years. Based on this reality, ageing with HIV is a newly manifested chronic disease with a complex long-term management in consideration also of the impact of HIV and HAART on the natural history of other chronic diseases typically associated with older age.CDC Centers for Diseases ControlHAART Highly Active Antiretroviral TherapyNRTI Nucleoside Reverse Trascriptase InhibitorNNRTI Non Nucleoside Reverse Trascriptase InhibitorPI Protease InhibitorVIS Viro Immunological SuccessVS Virological SuccessIS Immunological SuccessThe author(s) declare that they have no competing interests.MT conceived the study, performed the statistical analysis and wrote the manuscriptRR participated in the design of the study, in the collection of the data, in the statistical analysis and in the writing of the manuscriptKdGD participated in the design of the study, in the collection of the data and in the writing of the manuscriptSB participated in the design of the study and in the collection of the dataEM participated in the collection of the dataET participated in the collection of the dataET participated in the design of the studyRC did the supervision and critically read the last version of the paperAll authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:


To reduce the intolerable burden of malaria in pregnancy, the Ministry of Health in Tanzania has recently adopted a policy of intermittent presumptive treatment for pregnant women using sulphadoxine-pyrimethamine (IPTp-SP). In addition, there is strong national commitment to increase distribution of insecticide treated nets (ITNs) among pregnant women. This study explores the determinants of uptake for both ITNs and IPTp-SP by pregnant women and the role that individual knowledge and socio-economic status has to play for each.293 women were recruited post-partum at Kibaha District Hospital on the East African coast. The haemoglobin level of each woman was measured and a questionnaire administered.Use of both interventions was associated with a reduced risk of severe anaemia (Hb<8 g/dL) compared to women who had used neither intervention . In a logistic regression model it was found that attendance at MCH health education sessions was the only factor that predicted IPTp-SP use  while high knowledge of malaria predicted use of ITNs .Individual knowledge of malaria was an important factor for ITN uptake, but not for IPTp-SP use, which was reliant on delivery of information by MCH systems. When both these interventions were used, severe anaemia postpartum was reduced by 69% compared to use of neither, thus providing evidence of effectiveness of these interventions when used in combination. Plasmodium falciparum malaria in pregnancy poses a substantial risk to pregnant women and their offspring: it has been estimated that malaria in pregnancy is the primary cause of up to 10,000 maternal anaemia-related deaths in sub-Saharan Africa annually [annually .Over the last decade a body of evidence has accumulated which supports the use of both ITNs and IPTp-SP to reduce the adverse effects of malaria during pregnancy ,3,4 and Evidence suggests that malaria treatment choices are affected by knowledge of the problem ,8. The sThis research was carried out at Kibaha District Hospital in the Coastal Region of Tanzania, an area with moderate to high malaria transmission. The area is predominantly rural, supported by a mixture of subsistence and cash crop farming, with a peri-urban belt along the main Tanzania/Zambia highway which crosses the district.All post-natal women who had delivered their baby in the hospital during April/May 2003, but had not been admitted to hospital during the pregnancy, were eligible for inclusion in the study.©, HemoCue AB, Ängelholm, Sweden). Women with Hb<11 g/dL were referred within the hospital system.From studies elsewhere  it was eParticipants were assigned a 'knowledge of malaria score' (KoM) according to their responses to a series of seven closed questions Table .Women who reported that during this pregnancy they had normally (i.e. >75% of the time) slept under a bednet which had been impregnated within the previous six months.Use of intermittent presumptive treatment of malaria in pregnancy with sulfadoxine-pyrimethamine.Severe anaemia post-partum was defined as Hb<8 g/dL.2 P-value <0.10 or Mantel-Haenszel estimate of the rate ratio, P-value <0.10) were further analysed using multiple logistic regression. Significance in the multi-variate models was defined by a likelihood-ratio test (LRT) P-value <0.05.Data were entered twice in Epi-Info version 6.04 and analysed in Stata version 7 . Evidence of an association was sought between the variables listed in Table A completed questionnaire and measurement of haemoglobin level were available for 293 post-partum women whose characteristics are shown in Table The KoM assessment consisted of four parts: knowledge of risk, consequences of risk, transmission and prevention, with seven questions in total Table . Each quThe lowest knowledge scores related to the impact of maternal malaria on the health of the foetus; only 28% (81/293) recognized low birth weight and 38% (112/293) pregnancy loss as a potential consequence of maternal malaria. There was also some confusion over the mode of transmission of malaria: 95% (279/293) agreed that mosquitoes could transmit malaria but only 35% thought that mosquitoes alone were responsible (excepting blood transfusion).2 for linear trend 50.03, p < 0.0001). Because of colinearity between education level and high KoM, education was excluded from the logistic regression model used to identify determinants of high KoM. However, models for primary and secondary education respectively showed the same socio-economic effects as the data presented in Table Having high KoM was strongly associated with education level (χ48% (141/293) of women were ITN users, 57% (166/293) had received IPTp-SP once and 12% (34/293) IPTp-SP twice. A multi-variate analysis of factors influencing the uptake of these two interventions is shown in Table 41% (120/293) of women had used both interventions. Women with a median KoM were twice as likely (OR 2.1 (95% CI 1.1–4.0) and women with a high KoM three times more likely (OR 3.2 (95% CI 1.7–6.0) to have used both IPT-p and an ITN than women with low KoM scores.2 test for trend 5.1, p = 0.02).26% (76/293) of women had first attended the MCH during the first trimester, 65% (192/293) during the second trimester and 9% (25/293) during the third trimester of pregnancy. Predictably the median number of visits to MCH by women was correlated with the trimester of first visit: first trimester – median of 7.5 visits overall (mean 6.9 (s.d.2.2), second trimester – median of 5 visits overall (mean 5.4 (s.d.1.8) & third trimester – median of 2 visits overall (mean 2.4 (s.d.0.8). Women attending MCH for the first time during their first trimester were more than twice as likely to have attended health education sessions as women attending for the first time in their third trimester  of study participants had Hb<8 g/dL immediately post-partum. In the regression model, three factors were found to independently predict the risk of having severe anaemia in the post-partum period Table . FirstlyThe key finding was that while knowledge, wealth and age were all found to be independently predictive of ITN use, only participation in health education was associated with use of IPTp-SP. That predictors of uptake for each of the two interventions were different was an interesting and unexpected finding. The data indicate that, while ITN use is driven by both access  as well as knowledge, use of IPTp is determined by its being offered in an MCH clinic. This is a positive finding that suggests the need to prioritise strategies for maximising early attendance to boost IPTp-SP uptake.This finding is in contrast to research from Kenya which showed that uptake of IPTp-SP increased with higher levels of formal education . The NatThese findings on knowledge and awareness suggest that the increased risk posed to pregnant women by malaria was almost universally recognized, but that knowledge of the health impact of that risk – especially to the health of the foetus – was very low. Over 90% of women thought that ITNs were a good intervention against malaria in pregnancy, but less than half thought the same about IPTp-SP. Knowledge of malaria in pregnancy was strongly associated with use of a combination of both ITN and IPTp. Although this was a small observational study where it was not possible to control for all likely confounding variables, there is evidence that the use of a combination of ITN use and IPT-p provides additive protection against severe anaemia, and that this effect is not confined to trial conditions. Use of an ITN or IPT-p alone was associated with a 23% and 30% reduction in the risk of severe anaemia post partum respectively, similar to estimates from other controlled trials ,11 The dIt must be noted that by recruiting only women who had uncomplicated pregnancies and who delivered in hospital this study had a selection bias towards the healthier and possibly wealthy members of the community. However, women from a considerable range of socio-economic situations are represented  which probably reflects the rural/peri-urban catchment area of Kibaha and other newly urbanised areas. The data has shown that many of the factors indicative of relative wealth in a poor community were associated with increasing levels of knowledge – and that knowledge was positively associated with multiple intervention uptake and improved health outcome.The importance of access to resources has been illustrated previously for both preventative interventions and treatment . At the The findings highlight the importance of women's knowledge of malaria in pregnancy and of antenatal attendance for the uptake of preventative interventions. Now that effective malaria interventions are available and there is political will to implement them, to maximise the potential for health impact, it is essential to empower the intended recipients of interventions by providing the knowledge which can influence their health decisions.RN carried out the fieldwork and performed preliminary analysis. CD & HR contributed to study design and logistics. TM performed statistical analysis and supervised the study. All authors read, edited and approved the final manuscript.


It has been shown that the classical receptive fields of simple and complex cells in the primary visual cortex emerge from the statistical properties of natural images by forcing the cell responses to be maximally sparse or independent. We investigate how to learn features beyond the primary visual cortex from the statistical properties of modelled complex-cell outputs. In previous work, we showed that a new model, non-negative sparse coding, led to the emergence of features which code for contours of a given spatial frequency band.We applied ordinary independent component analysis to modelled outputs of complex cells that span different frequency bands. The analysis led to the emergence of features which pool spatially coherent across-frequency activity in the modelled primary visual cortex. Thus, the statistically optimal way of processing complex-cell outputs abandons separate frequency channels, while preserving and even enhancing orientation tuning and spatial localization. As a technical aside, we found that the non-negativity constraint is not necessary: ordinary independent component analysis produces essentially the same results as our previous work.We propose that the pooling that emerges allows the features to code for realistic low-level image features related to step edges. Further, the results prove the viability of statistical modelling of natural images as a framework that produces quantitative predictions of visual processing. A number of models approach the computational modelling of primary visual cortex by using two processing stages. First, there is a linear filtering with filters that are bandpass, oriented, and spatially localized. In some models, the outputs of the linear filters are half-wave rectified, but this difference is inessential because a rectification is done in the second stage anyway. The second stage then consists of pooling together rectified outputs of the first stage, so that cells that have the same orientation and frequency, as well as similar spatial locations, are pooled together. This pooling is then essentially a summation of rectified outputs of filters of different phases. These two processing steps are assumed to roughly correspond to simple and complex cells in V1, respectively. While there is controversy of the validity of such models, see e.g. , this isRecent research has seen a number of models that attempt to explain these processing stages based on statistical modelling of natural images . First, application of independent component analysis (ICA)  or sparspredictive manner, so that we would be able to predict properties of cells in the visual cortex, in cases where the properties have not yet been demonstrated experimentally. This would give testable, quantitative hypotheses that might lead to great advances, especially in the research in extrastriate areas such as V2, whose function is not well understood at this point.It would be most useful if we could use this modelling endeavour in a Here, we attempt to accomplish such predictive modelling in order to predict properties of a third processing step, following the two described above. Previously, we have applied a modification of the ICA / sparse coding model on the outputs of modelled complex cells whose input consisted of natural images . The modWe extend our previous results in two ways. The complex cells in our previous work were all constrained to have the same frequency, which was done in order to reduce the computational load. Here, we first report a technical advance: it is not necessary to make the assumptions of nonnegativity as in . Thus, wAs described in Methods, we input a large number of natural image patches into model complex cells that computed the sum of squares of outputs of two simple cells, one odd-symmetric and the other even-symmetric. Then, we performed independent component analysis of the complex cell outputs using the FastICA algorithm.f2 in Figure In the first experiment, we used only the output from complex cells in a single frequency band, ai which show the contribution of the third-stage feature of index i on the activities of complex cells. A collection of the obtained basis vectors is shown in Figure g1  seems to lead to the largest number of long contours.Other FastICA nonlinearities led to similar basis vectors. However, some led to a larger number of longer contours. Figure ai with corresponding activities si.In the second experiment, the complex-cell set was expanded to include cells of three different preferred frequencies. In total, there were now 432 complex cells. We performed ICA on the complex-cell outputs when their input consisted of natural images. Thus, we obtained 432 higher-order basis vectors (features) We visualized a random selection of higher-order features learned from natural images in Figure Pi in Equation (4) of Methods for the learned basis vectors. The distribution of this measure for natural image input and white Gaussian noise input is shown in Figure Pi that was larger than this quantile. (For the 95% quantile the proportion was 63%.) Thus, we can say that more than half of the higher-order basis vectors, when learned from natural images, have a pooling over frequencies that is significantly above chance level.We computed the frequency pooling measure Pi above its 99% quantile for white Gaussian noise). These are plotted in Figure To show that the pooling measure is valid, and to further visualize the frequency pooling in the higher-order features, we chose randomly basis vectors learned from natural images that have pooling significantly over chance level *  and tuned to the preferred orientation of the higher-order feature for every frequency f. This shows how the higher-order features are largely selective to a single orientation. When the input consisted of Gaussian white noise, only 34% of the cells were found to be orientation-selective according to this criterion.We also examined quantitatively whether the higher-order features are tuned to orientation. We investigated which complex cell has the maximum weight in Finally, we synthesized images from higher-order feature activities to further visualize the higher-order features (see Methods). Figure What is the functional meaning of the pooling we have found? We propose that this spatially coherent pooling of multiple frequencies leads to representation of an edge that is more realistic than the band-pass edges given by typical Gabor filters . PresumaIn terms of frequency channels, the model predicts that frequency channels should be pooled together after complex cell processing. Models based on frequency channels and related concepts have been most prominent in image coding literature in recent years, both in biological and computer vision circles. The utility of frequency channels in the initial processing stages is widely acknowledged, and it is not put into question by our results – in fact, the statistical modelling framework does show that using band-pass simple and complex cells is statistically optimal ,13. HoweSeveral investigators have looked at the connection between natural image statistics, Gestalt grouping rules, and local interactions in the visual cortex ,19-21. Hsi and the coefficients aki were constrained to be non-negative, following proposals by [Compared to our own previous work , the maiosals by ,24. HoweRecent measurements from cat area 18  emphasize responses to "second-order" or "non-Fourier" stimuli, typically sine-wave gratings whose amplitudes are modulated ,25. ThesAnother closely related line of work is by Zetzsche and coworkers ,29 who eThe present results are an instance of predictive modelling, where we attempt to predict properties of cells and cell assemblies that have not yet been observed in experiments. To be precise, the prediction is that in V2 (or some related area) there should be cells whose optimal stimulus is a broad-band edge that has no sidelobes while being relatively sharp, i.e. the optimal stimulus is closer to a step-edge than the band-pass edges that tend to be optimal for V1 simple and complex cells. The optimal stimulus should also be more elongated ,31 than Statistical models of natural images offer a framework that lends itself to predictive modelling of the visual cortex. First, they offer a framework where we often see emergence of new kinds of feature detectors – sometimes very different from what was expected when the model was formulated. Second, the framework is highly constrained and data-driven. The rigorous theory of statistical estimation makes it rather difficult to insert the theorist's subjective expectations in the model, and therefore the results are strongly determined by the data. Third, the framework is very constructive. From just a couple of simple theoretical specifications, e.g. non-Gaussianity, natural images lead to the emergence of complex phenomena.We hope that the present work as well as future results in the same direction will serve as a basis for a new kind of synergy between theoretical and experimental neuroscience.We have shown that pooling over complex cells of different frequency preferences emerges when we model the statistical properties of natural images. This is accomplished by applying ordinary ICA on a set of modelled complex cells with multiple frequencies, and inputting natural images to the complex cells. The resulting independent components, as represented by the corresponding basis vectors, code for simultaneous activation of complex cells that have similar orientations, form a collinear configuration, and span multiple frequencies. Thus, statistical modelling of natural stimuli leads to an interesting hypothesis on the existence of a new kind of cells in the visual cortex. (category "deblurred") [I.The natural images were 1008 gray-scale images of size 1024 × 1536 pixels from van Hateren's database, available at xk to natural images were modelled with a classical energy model:The complex-cell model was similar to our previous work . The fil and  are even- and odd-symmetric Gabor receptive fields whose energies are pooled together in the complex cell. The complex cells had 6 × 6 = 36 different spatial locations, and at each location, four different preferred orientations and three different frequency bands. The aspect ratio was fixed to 1.5 and frequency bandwidth to 1.5 octaves, which implied an orientation bandwidth of 37°, according to the definitions in [W were normalized to unit norm for visualization purposes. The actual normalization we used in the experiments consisted of standardizing the variances of the complex cell outputs so that they were equal to unity for natural image input. The number of complex cells totalled K = 36 × 4 × 3 = 432. Note, however, that in Experiment 1 we only used a single frequency band.where tions in . The frex =  using the FastICA algorithm [g were used, see Table Independent component analysis (ICA) was performed on the vector lgorithm . The ortx = x,...,xK uor in vector formai =  gives a higher-order basis vector. The si define the values of the higher-order features in the third cortical processing stage.where the vector ai and -ai because any change in sign of the basis vector can be cancelled by changing the sign of si accordingly. Here, we defined the sign for each vector ai so that the sign of the element with the maximal absolute value was positive.Note that the signs of the basis vectors are not defined by the ICA model , i.e. thTo obtain a baseline with which to compare our results, and to show which part of the results is due to the statistical properties of natural images instead of some intrinsic properties of our filterbank and analysis methods, we did exactly the same kind of analysis for 24 × 24 image patches that consisted of white Gaussian noise, i.e. the gray-scale value in each pixel was randomly and independently drawn from a Gaussian distribution of zero mean and unit variance. The white Gaussian noise input provides a "chance level" for any quantities computed from the ICA results.ai following [k, and the brightness of the ellipse is proportional to the aki coefficient of the basis vector ai, using a gray-scale coding of coefficient values. In Experiment 1, i.e. the case with a single frequency band, we used this method directly to visualize each higher-order basis vector in a single display. In Experiment 2, i.e. the multifrequency case, we visualized each frequency band separately.We visualized the resulting higher-order basis vectors ollowing  by plottai the coefficient in the higher-order basis vector ai that corresponds to the complex cell with spatial location , orientation θ and preferred frequency fn. We computed a quantity which is similar to the sums of correlations of the coefficients over the three frequency bands, but normalized in a slightly different way. This measure Pi was defined as follows:In Experiment 2, we are interested in the frequency pooling of complex cells in different higher-order features. We quantified the pooling over frequencies using a simple measure defined as follows. Let us denote by Cm is defined aswhere the normalization constant Cn.and likewise for i the hot-spot * as the centre location  of complex cells where the higher-order component si generates the maximum amount of activity. That is, we sum the elements of ai that correspond to a single spatial location, and choose the largest sum. This allows us to define the tuning to a given orientation of a higher-order feature i by summing over the elements of ai that correspond to the spatial hotspot and a given orientation; the preferred orientation is the orientation for which this sum is maximized. We also computed the length of a higher-order feature as described in [For further analysis of the estimated basis vectors, we defined the preferred orientation of a higher-order feature. First, let us define for a higher-order feature of index ribed in .W in simple cells are equal to the corresponding basis vectors, and that all the elements in the higher-order basis vector are non-negative . Then, the synthesized image  for higher-order basis vector ai is given byIt is also possible to perform an image synthesis from a higher-order basis vector. However, the mapping from image to complex-cell outputs is not one-to-one. This means that the generation of the image is not uniquely defined given the activities of higher-order features alone. A unique definition can be achieved by constraining the phases of the complex cells. We assume that only odd-symmetric Gabor filters are active. Furthermore, we make the simplifying assumptions that the receptive fields H denotes the set of indices that correspond to complex cells of the preferred orientation at the hotspot. Negative values of aki were set to zero in this synthesis formula.where the square root cancels the squaring operation in the computation of complex-cell responses, and A.H. conceived the basic idea and the principles of the experimental set-up, and wrote the paper. M.G. performed the experiments and elaborated the experimental set-up. P.O.H. assisted in the experiments and the writing.