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TITLE: Effects of Neuromuscular Electrical Stimulation Combined with Exercises versus an Exercise Program on the Pain and the Function in Patients with Knee Osteoarthritis: A Randomized Controlled Trial ABSTRACT: Objectives. To investigate the effect of 8 weeks of NMES + Ex (neuromuscular electrical stimulation combined with exercises) on pain and functional improvement in patients with knee osteoarthritis (OA) compared to exercise (Ex) alone. Design. Randomized controlled trial. Setting. A specialty outpatient clinic. Participants. Patients (N = 100; women = 86, men = 14; age range, 50–75 years) with knee OA. Interventions. Participants were randomly assigned to NMES + Ex or Ex group. Outcome Measures. Numerical Rating Scale 0 to 10 (NRS) and the Timed Up and Go (TUG) test were the primary outcomes. The secondary outcomes used were the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). Results. Following the interventions, a statistically significant improvement in both groups was observed in all outcomes assessed. For the comparison between the groups, no statistically significant difference was found between the NMES + Ex and the Ex groups in NRS (P = 0.52), TUG test (P = 0.12), and aspects of WOMAC: pain (P = 0.26), function (P = 0.23), and stiffness (P = 0.63). Conclusion. The addition of NMES to exercise did not improve the outcomes assessed in knee OA patients. This study was registered at the Australian Clinical Trials Registry (ACTRN012607000357459). BODY.1. INTRODUCTION: Osteoarthritis (OA) is the most common form of arthritis. It affects one-third of adults and tends to increase with age [1]. Knee OA is associated with symptoms of pain, swelling, instability, and reduced range of motion (ROM). These symptoms lead to functional impairment, increasing the risk of morbidity and mortality [2, 3]. The synovium is infiltrated with inflammatory cells and secretes excess synovial fluid, leading to capsular swelling [4]. Through a spinal reflex, the capsular swelling inhibits muscle activation, which, combined with disuse, may cause muscle weakness and atrophy [1]. Because the quadriceps muscle acts as shock absorber for the knee joint, weakness in the thigh muscle reduces joint protection, resulting in overload [4]. Exercises strengthen the muscles, reduce pain, improve physical function, and are therefore considered a major intervention in the conservative treatment of patients with knee OA [5]. In addition to muscle strengthening exercises, stretching exercises are commonly used to increase ROM and are often prescribed in rehabilitation protocols as part of routine warm-up to prepare the muscles and joints for other types of exercise, such as aerobic and strengthening programs [6, 7]. Stretching of the hamstring muscles may improve knee extension ROM in OA patients. Neuromuscular electrical stimulation (NMES) is defined as the application of electrical stimulation using surface electrodes placed over skeletal muscles to produce visible muscle contraction through the activation of intramuscular nerve branches [8]. This technique can also be used as a form of physical therapy in the treatment of patients with knee OA. The goals of rehabilitation protocols that include NMES are to provide additional stimulus to increase muscle strength in patients with knee OA [9]. The methods and findings of previous studies on the effectiveness of NMES in knee OA differ in the modulation of NMES parameters, choice of the outcomes used to evaluate the patients, and characteristics of the control groups. This leads to a lack of consensus regarding the effectiveness attained from including NMES in conventional rehabilitation protocols. Major flaws with regard to methodological quality were found in clinical trials testing the use of NMES in the conservative treatment of patients with knee OA. Only one of these studies reported using allocation concealment, blinded assessment, and intention-to-treat analysis [10]. Thus, the objective of this study was to conduct a randomized clinical trial following methodological criteria, including allocation concealment, blinding of the examiner, and application of intention-to-treat analysis to assess the role of NMES in improving pain and physical function in patients with knee OA. It is important to study interventions with the potential to improve functional status in this patient population [11]. BODY.2. METHODS: The study was conducted at the Interlagos Specialty Outpatient Clinic, São Paulo, Brazil. Patients were referred from the Rheumatology Department according to the inclusion and exclusion criteria and randomly allocated into groups using a computer-generated randomization chart. The allocation codes were sealed in opaque envelopes by a third person not involved in the study to avoid selection bias. Written informed consent was obtained from all participants. The study was approved by the Research Ethics Committee of the Universidade Federal de São Paulo (UNIFESP), Brazil, no. 0141/07, and registered with the Australian Clinical Trials Registry, no. ACTRN012607000357459. Setting the significance level at 5% and the power of the sample at 80%, a sample size of 40 patients per group was estimated to be necessary to detect a difference of at least 1 minute ± 3 seconds in the Timed Up and Go (TUG) test, which was considered to be the minimum clinically significant difference for the present trial [12]. Paired Student's t-test and analysis of covariance (ANCOVA) were used for comparisons between groups; the covariant was obtained from a previous study [13]. One hundred patients were recruited according to inclusion and exclusion criteria. Eligibility criteria were age 50 to 75 years, OA grade 2 or greater according to the radiographic classification of OA proposed by Kellgren and Lawrence [14], and diagnosis of knee OA based on the American College of Rheumatology (ACR) criteria. Exclusion criteria were use of a pacemaker, unstable heart conditions, participation in another physical activity program, inability to exercise on a stationary bicycle ergometer, inability to walk, previous hip or knee arthroplasty, diagnosis of fibromyalgia, epilepsy, and skin tumor or lesion at the NMES application site. Patients were divided into two groups of 50 each: (1) the NMES combined with exercises (NMES + Ex) group and (2) exercise (Ex) group. Patient medication was standardized and not modified during the study period. Paracetamol was prescribed for pain, and diacerein and chloroquine for OA control. Interventions were delivered for both groups by the same physical therapist, twice a week, for 8 weeks, with each session lasting about 40 minutes. All patients received a manual including guidelines on how not to overload the knee during daily activities and instructions on the use of ice packs in case of pain and inflammation and warm compresses in case of pain without inflammation as follows. Manual for Patients with Osteoarthritis of the Knee. The purpose of this manual is to explain osteoarthritis and to teach how you can adjust yourself to your daily activities, according to the knee symptoms. Try to seriously follow our orientations for your own benefit! The Knee. The knee joint is composed of 3 bones—the femur (thigh bone), the patella (kneecap) and the tibia (leg bone). It has muscles, capsule, ligaments, meniscus, and the cartilage that lines the bones and protects them from the impact. The knee joint supports nearly the whole weight of our body. What Is Osteoarthritis? It is a disease caused by the breakdown of cartilage in the joints. The layers in the cartilage become damaged and with time they lose the function of smoothing the contact between the bone surface and the joints. The pain is a result of the attrition of one bone against the other in the absence or decreased cartilage in the joints. What Are the Signs and Symptoms? Patients with osteoarthritis may have some pain mainly when starting a movement, as in morning stiffness or after immobilization. With time, the pain might be intensified and be permanent. The presence of crepitation when moving the knees is often. What Kind of Difficulties Might I Have in My Daily Life? Difficulties found in daily live vary according to the patients' symptoms. In general, however, the patient has pain and difficulty when supporting the body weight using the affected knee, going up and down the stairs, or when walking. What Should I Do When It Is Painful? A doctor can prescribe the treatment for osteoarthritis. However, a simple form of improving the pain is to use warm to hot water bottle over the knee joint (be careful not to burn the skin, use a protection, and test the water temperature before using it. What If It Is Swollen? To manage the swollen, you can combine rest, use of ice pack, and elevating the leg above the level of the heart. The ice pack should be placed over the knee joint for 20 minutes. What Are the Other Recommendations? If you are overweight, losing some kilos will reduce the stress over the joint. Wear comfortable shoes with a rubber sole and no heels.In case of pain when walking, use a cane as an aid tool.Try to have a good night sleep. BODY.2. METHODS.2.1. NMES COMBINED WITH EXERCISE GROUP: Treatment for patients in the NMES + Ex included 10 minutes on a stationary bicycle, stretching of hamstring muscles (3 repetitions of 30 seconds) with the aid of an elastic band, and loaded quadriceps strengthening exercises combined with NMES. The strengthening exercise with NMES was performed in the sitting position with the knee and hip flexed to 90 degrees; patients contracted their quadriceps at each NMES stimulus. NMES was applied using an electrical stimulator (Globus ACTIVA 600 Pro, Globus, Italia) with two 7.5 × 13 cm self-adhesive electrodes (ValuTrode electrodes, Axelgaard Manufacturing Co. Ltd., Fallbrook, CA) placed over the region of the quadriceps muscle (rectus femoris and vastus medialis). NMES parameters were as follows: pulsed current, biphasic, asymmetrical, rectangular waveform, frequency 50 Hz, pulse duration 250 μs, contraction time 10 s, rest time 30 s every 20 minutes; current intensity was the maximum tolerated by each patient [15]. BODY.2. METHODS.2.2. EXERCISE GROUP (EX): Patients in the Ex group performed the same exercise program as those in the NMES group but without NMES. The exercise protocol included 10 minutes of warm-up on a stationary bicycle ergometer, stretching of hamstrings muscles with the aid of an elastic band, and knee extension exercises performed for 3 sets of 15 repetitions with rest intervals of 30–45 seconds between sets. For both groups, the training load for the strengthening exercises was established based on 50–60% of the 10-repetition maximum (RM) instead of 1 RM to avoid injury by excessive muscle contraction [16]. BODY.2. METHODS.2.3. OUTCOMES: Patients were evaluated before and after intervention by a physical therapist blinded to group assignment. The primary outcomes were the TUG test results [12] and pain walking on a flat surface in the last 72 h measured on an 11-point Numerical Rating Scale (NRS) [17]. The secondary outcomes were scores on the pain, physical function, and stiffness subscales of the culturally validated Brazilian-Portuguese version of the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) [18, 19]. In this study, WOMAC pain, physical function, and stiffness scores were analyzed separately. BODY.2. METHODS.2.4. STATISTICAL ANALYSIS: Paired Student's t-test was performed to compare pre- and postintervention values at a significance level of 0.05 (P < 0.05). Statistical analysis was performed on an intent-to-treat (ITT) basis and included all patients who were randomized to treatment. Mixed model analysis of variance with repeated measures was used with occasion measures as within-group factors and intervention as between-group factor. Relations between the observations were analyzed using an unstructured covariance matrix. Missing-data imputation was not performed to evaluate pre- and postintervention differences between the two groups, because Chakraborty and GU [20] showed that mixed model analysis without missing-data imputation always provides equal or more power than does mixed model analysis with missing-data imputation. Effect size was calculated as the difference between the means divided by the standard deviation using Cohen's d [21]. The analyses were performed using the General Linear Model (GLM), and mixed analyses were carried out using the Statistical Analysis Software (SAS) version 9.2 for Windows [22]. BODY.3. RESULTS: The demographic and clinical characteristics of patients, including age, sex, side treated, and body mass index (BMI), as well as TUG test values, NRS pain scores, and WOMAC scores on the pain, physical function, and stiffness subscales, are shown in Table 1. Eighty-seven patients completed the study. Of the 13 dropouts, 6 (12%) patients were in the NMES + Ex group and 7 (14%) in the Ex group (Figure 1). BODY.3. RESULTS.3.1. PRIMARY OUTCOMES: No significant differences between groups were found in NRS pain scores and TUG test time on ITT analysis. A significant decrease in pain intensity (NRS scores) and TUG test time was observed after intervention compared with baseline in the NMES + Ex group (NRS scores, P < 0.0001; TUG test, P < 0.0001) and Ex group (NRS scores, P < 0.0001; TUG test, P < 0.0001). The primary outcomes are listed in Table 2. BODY.3. RESULTS.3.2. SECONDARY OUTCOMES: No significant differences between groups were found on the pain, physical function, and stiffness subscales of the WOMAC index on ITT analysis. There was a significant improvement in all WOMAC subscales in the NMES + Ex group (pain; P < 0.0001; physical function, P < 0.0001; stiffness, P < 0.0001) and in the Ex group (pain, P < 0.0001; physical function, P < 0.0001; stiffness, P = 0.0009). The secondary outcomes are listed in Table 2. BODY.3. RESULTS.3.3. ADVERSE EFFECT: One patient in the NMES + Ex group exhibited a blood pressure spike, which may have resulted from the use of NMES or from the exercise program itself. The following contraindications to the use of NMES were respected: avoiding the use of NMES over areas of tumor, with open wounds, or bleeding, and in patients with pacemakers [15]. BODY.4. DISCUSSION: In this randomized clinical trial, the NMES combined with exercise did improve pain and physical function, but there was no evidence that it did better than exercise alone. Our results are in agreement with the findings of Rosemffet et al. [23] who conducted a pilot study comparing the treatment results of patients with knee OA (n = 26) treated with either NMES alone or exercises alone or exercises combined with NMES. The authors reported a significant improvement on the WOMAC pain subscale in the three groups, but no significant differences in WOMAC scores were found between groups [23]. The lack of difference in treatment outcome between groups in the present study might be attributed to the fact that the participants had no clinically significant muscle or functional impairment. A finding substantiating this hypothesis is that the mean TUG test values in the two groups were similar to those found in the study of Steffen et al., for elderly patients with no physical limitations categorized under the same mean age group [24]. The TUG test values reported were 8 ± 2 seconds for patients with 60–69 years old and 9 ± 3 seconds for patients with 70–79 years old. In the present study, the baseline values for TUG test were 8.27 + 1.76 for NMES + Ex group and 9.34 ± 2.47 for Ex group. Given that the greater the muscle impairment, the greater the NMES effect, patients with a more advanced stage of OA might obtain greater benefit from NMES [25–27]. With regard to evidence on the effectiveness of NMES in the rehabilitation of patients with knee OA, the Cochrane Collaboration published a systematic review evaluating the pre- and postoperative use of NMES in total knee arthroplasty [28]. The two studies that were evaluated were classified as being at high risk of bias, because randomization and allocation concealment were not described, and no data obtained from the study groups were presented in terms of means and standard deviations. It was not possible to determine whether or not the pre- and postoperative use of NMES was effective in the rehabilitation of knee arthroplasty patients, and therefore further studies are warranted. Regarding lines of evidence on the effectiveness of NMES in the treatment of other conditions, Kim et al. [29] conducted a review of the use of NMES after anterior cruciate ligament (ACL) reconstruction. Based on eight different studies, the authors concluded that the use of NMES combined with exercises can be effective in improving quadriceps strength up to four weeks after surgery. However, there was no sufficient evidence to affirm that NMES has any positive effect on the functional performance of patients who had undergone ACL reconstruction [29]. No literature review could be found on the effectiveness of NMES in patients with knee OA without indication for knee arthroplasty. A limitation of this study is that the current intensity used for electrical stimulation was not recorded. However, the maximum current intensity tolerated by each patient was applied as in previous studies [9, 24, 30]. No participant showed intolerance to electrical stimulation. The questionnaires used in this study have been translated into Brazilian Portuguese, cross-culturally adapted, and validated in previous studies [19, 31, 32]. The assessment of patient-reported pain intensity and physical function was performed according to the international consensus on outcomes measures for phase III clinical trials in OA, in which pain and physical function are identified as the most important outcome measures in randomized clinical trials [18]. The TUG test was chosen because it is a simple and inexpensive test designed to assess the functional mobility of patients based on activities of daily living [12]. Statistical analysis was performed on an intent-to-treat basis to minimize the impact of protocol violations (which may occur after randomization) on the results and conclusions and to avoid an overestimation of the treatment effect. This randomized clinical trial conforms to the Consort Statement (Consolidated Standards of Reporting Trials) [33], whose aim is to improve the quality of reports of randomized clinical trials. Given the predominance of mild and moderate OA cases among the participants, the results of this study can be generalized especially for patients with similar severity levels of OA. BODY.5. CONCLUSIONS: Our results revealed that the application of NMES combined with a conventional exercise program was as effective as the exercise program alone in reducing pain and improving physical function in patients with knee OA, and therefore no therapeutic benefit was observed with the use of NMES. BODY.6. CLINICAL IMPLICATIONS: Moderate exercises, including warm-up and muscle stretching and strengthening exercises—combined or not with NMES—are recommended to reduce pain and improve physical function and quality of life in patients with knee OA.

TITLE: The Effect of Breathing Exercises on the Nocturnal Enuresis in the Children with the Sleep-Disordered Breathing ABSTRACT.BACKGROUND: The nocturnal enuresis is one of the most common complaints of childhood. Upper airway obstruction and nocturnal snoring affect the nocturnal enuresis in children. ABSTRACT.OBJECTIVES: The aim of this study was to investigate the effects of breathing exercises on the nocturnal enuresis in the children with the sleep-disordered breathing. ABSTRACT.PATIENTS AND METHODS: This study was conducted in year of 2011 by a semi-experimental design with the control group among 40 children, aged 6 - 12 years, who had the nocturnal enuresis. Participants were examined based on the criteria of nocturnal enuresis, oral breathing, and nocturnal snoring. Subsequently, they were randomly assigned to the case and control groups. In the case group, the breathing exercises were performed for 45 minutes, and were pursued for four weeks in the morning following and prior to sleeping, and subsequently the arterial blood gases were measured and the frequency of enuresis and the respiratory rates (RR) were recorded. ABSTRACT.RESULTS: After intervention the means of PaCO2 and RR in the control group were significantly higher than the case group (P < 0.0001). Likewise, O2sat, PaO2 in the case group were higher than the control group (P < 0.0001). The nocturnal enuresis decreased significantly in the case group, compared to the control group (P < 0.0001). ABSTRACT.CONCLUSIONS: This study suggests that the breathing exercises may reduce the frequency of nocturnal enuresis in the patients with the oral breathing and nocturnal snore. The clinical implications of these findings should be verified in the future longitudinal studies. BODY.1. BACKGROUND: Nocturnal enuresis is defined an accidental urination with at least, twice per week for three consecutive months, and calendar age of five years old (1). It is one of the most common complaints of childhood; children from the age of five should be able to control the urine (2, 3) because at this age, bladder capacity increases and brain nerve centers control the urinary bladder contractions during sleep (4). Many children cannot reach this stage. Therefore, children and their families face severe behavioral and social problems (1). Various methods have been proposed for the treatment of nocturnal enuresis, none of which is of a definitive efficacy (2, 3, 5). One of the findings concerning this disorder is that the nocturnal enuresis in children with the sleep-disordered breathing, whose nostrils are blocked, happens more (6-8). Relaxed muscles which help to open the airways and soft palate vibrations during sleep, will cause snoring, while in the normal condition air enters through the nose during the aspiration and slip on the edge of the soft palate. If this little edge moves and makes a barrier to the air, the sound of snoring is created (8, 9). Chang et al. (10) and Yeung et al. (11) have shown that the prevalence of nocturnal enuresis in children with the habitual snoring is four times more and 46 percent of children with the sleep- disordered breathing syndrome have the nocturnal enuresis (12). In some children, recurrent upper respiratory tract persistent infections, adenoids, habitual oral breathing, blocked Eustachian tube and nocturnal snoring can cause enuresis (13, 14). Children's physical health is dependent on how they are breathing. The natural activity of respiratory tract is controlled through the neck muscles, inter-rib muscles, abdominal muscles, and diaphragm. Some of the breathing disorders occur when the transferred messages through the brain are unable to transfer to these muscles through the spinal cord (15, 16). In children with the oral breathing, the superficial and shallow breathing create the nocturnal snore due to increasing weakness of breathing muscles. This factor causes the bladder to lose its control on urination (17, 18). Ezzat waleeda et al. have shown that opening blocked airway by the surgery will reduce the nocturnal enuresis in the children with the respiratory disorders (16). Breath control is done by the aspiration and expiration. In breathing exercises, the individual capacity will increase and the equilibrium is established between the nervous and respiratory systems and the mind will work normally (19), so that by the deep breathing, the peripheral air amount will also increase substantially. Respiratory exercises, tolerance exercises and exercise activities related to the upper and lower parts improve breathing process and create a balanced ventilation system (20). The reason is that individuals under the influence of respiratory exercises show an increase in the lung functional indices, and thus, their oxygen consumption volume rises (18, 21). A few external studies are present concerning the effect of breathing exercises improvement on the children nocturnal enuresis with the conflicting results, but we were unable to find any internal reference in this regard (15, 17, 22). BODY.2. OBJECTIVES: Therefore, the aim of this study was to investigate the effects of breathing exercises on the reduction of the nocturnal enuresis in the children with the sleep-disordered breathing. BODY.3. PATIENTS AND METHODS.3.1. PATIENTS: This study was conducted in year of 2011 by a semi-experimental study with a control group. The control and case groups were selected based on the DSM-IV-TR criteria by the convenience sampling (23). The groups were children, who had the nocturnal enuresis, and hence; were brought to the kidney and urethra clinic affiliated to the Isfahan University of Medical Sciences by their parents. The children's problem of breathing was also diagnosed by the ear, nose and throat specialists. Both groups were consisted of 40 children (6-12 years old) with the concomitant nocturnal enuresis and oral breathing. The 40 children were classified into the two equal groups. Participants were examined based on the criteria of oral breathing, nocturnal snoring, and nocturnal enuresis two times per week and the sleep-disordered breathing confirmed by the otolaryngologists. The exclusion criteria consisted of the current drug consumption (as, tetracycline, nitrofurantoin, methicillin, acethazolamide may decrease the level of PaCO2, whereas bicarbonate, hydrocortisone, metolazone may increase the level of PaO2), (24) mental and behavioral disorders, tonsillitis, physical disability, mental retardation, allergies and cigarette smoking by the parents. BODY.3. PATIENTS AND METHODS.3.2. PROCEDURE: First nocturnal enuresis frequency and the RR were recorded in the case and control groups. The RR was measured when the children were at rest and involved counting the number of breaths for one minute by counting how many times the chest rose. In addition, the patients of both groups were analyzed for the blood arterial gases. Subsequently, breathing exercises were taught to the children and their parents in the case group by the demo games and video clips by speech therapist. The patients did not drink water/any other liquids two hours before sleep. Then breathing exercises pursued in four weeks in the morning after getting-up, and also at night before sleep. Firstly, the breathing exercises of hip, anal and neck muscles and diaphragm were performed. For the evacuation of abdominal breathing toward the spine, diaphragm was drawn to the ribs. Then, the movements of the head and spinal column spin the effective energy in the body. During these exercises, breathing was done by the right nostril. Then, breathing exercises of the nose and jaw were done with closing the right nostril and pushing the chin to the chest, while expiration was done by the left nostril. Finally, during abdominal exercises, the inspiration was retained and with mouth closing, expiration expelled through the right nostril (14, 23). Then, the frequency of nocturnal enuresis and the RR were recorded. Upon the completion of breathing exercises, the arterial blood gases were also measured. BODY.3. PATIENTS AND METHODS.3.3. COLLECTION AND HANDLING OF BLOOD SAMPLES: A radial arterial blood of five milliliters was drawn fasting by the air-free bubbles plastic syringes (gauge 23) containing lyophilized heparin. Subsequently, the blood samples under the condition of air-sealed were placed on the ice bag instantly, and sent in a pre-cold chamber (approximate temperature 4 ̊C) to the laboratory within 10 minutes for the blood gases analysis. The laboratory also carried out the assay within 30 minutes (24). The arterial blood gas analyzer (ABL 300, Radiometer, Copenhagen, Denmark) was employed to measure the oxygen, carbon dioxide and pH levels. BODY.3. PATIENTS AND METHODS.3.4. STATISTICAL ANALYSIS: Statistical analyses were performed using SPSS software, version 16.0. Data presented as mean ± SD, median [IQR] and number as appropriate. At the baseline age, weight, height, body mass index (BMI), O2sat, PaO2, PaCO2, pH and RR were compared before and after the study in the control and case groups, using the independent, paired t-tests, and repeated measurements of ANOVA. In addition, the Chi-Square test was used to assess the distribution of sex between the groups. Likewise, nocturnal enuresis frequency was compared, before and after the study within and between the groups, using the Wilcoxon Signed Rank and Mann-Whitney tests, respectively. Statistical significance was accepted at the p-value less than 0.05. BODY.3. PATIENTS AND METHODS.3.5. ETHICAL CONSIDERATION: This study was approved by the Ethics Committee of the Isfahan University of Medical Sciences. Written informed consent was obtained from the parents, and that the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priory approval by the institution's human research committee. BODY.4. RESULTS: Table 1 displays the total number of the case group consisted of three girls and 17 boys. The case group was between 6 to 10 years old, with a mean age of 7.09 ± 1.23. In addition, the control group was between 6 to 11 years old, with a mean age of 8.12 ± 1.74. The results showed that there was insignificant difference in gender between the two groups. In addition, there was insignificant difference in the BMI, weight, and height between the case and the control groups. Table 1. Demographic Characteristics of the Groups. Case group a Control group a P value Weight, kg 29.5 (2.81) 29.3 (2.16) 0.824 b Height, cm 127.4 (2.19) 126.9 (2.04) 0.945 b BMI, kg/m2 15.92 (2.31) 16.04 (1.08) 0.129 b Age 6-11 7.09 (1.23) 8.12 (1.74) 0.421 c Gender Male 3 6 Female 17 14 0.189 b a Values are represented as mean (SD) b P values were calculated by the Chi Squared c P values were calculated by the independent t-test Table 2 displays the comparison of the means of the O 2 sat, PaO 2 , PaCO 2 , pH and RR between the groups before and after intervention. Before intervention, the means of O 2 sat, PaO 2 , PaCO 2 , pH and RR were similar in both groups (P > 0.05). After intervention, only pH was similar in both groups (P > 0.05) and the means of O 2 sat, PaO 2 in the case group were significantly higher than the control group (P < 0.0001). However, in the control group, the means of the PaCO 2 and RR were significantly higher than the case group (P < 0.0001). Table 2. Comparison of the O 2 sat, PaO 2 , PaCO 2 , pH and RR between the Groups. Control Group a Case Group a P value b Before After Before After O 2 sat, mm/Hg c 84.61 (1.13) 84.26 (1.21) 83.92 (3.29) 89.45 (2.17) 0.001 P value d 0.92 0.01 PaO, mm/Hg 2 , mm/Hg e 69.54 (1.26) 67.20 (2.14) 75.28 (2.43) 0.001 P value d 0.091 0.001 PaCO 2 , mm/Hg 75.16 (1.56) 76.12 (1.23) 72.30 (1.49) 60.21 (1.28) 0.001 P value d 0.34 0.001 pH, mm/Hg 7.76 (1.34) 7.43 (1.23) 7.91 (2.28) 7.30 (2.19) 0.124 P value d 0.87 0.12 RR f 29.61 (1.21) 29.12 (1.61) 27.42 (1.06) 21.34 (1.16) 0.001 P value d 0.91 0.01 a Values are represented as mean (SD) b P-values were calculated by the repeated measurements of ANOVA c P-values were calculated by the Oxygen 2 saturation d P-values were calculated by the paired t-test. e P-values were calculated by the partial pressure of O 2 f P-values were calculated by the Respiratory Rates Figure 1.Depicts the Comparison of the Means of the O 2 sat, PaO 2 , PaCO 2 , pH and RR Between the Groups Before and After Intervention.Before intervention, there were insignificant alteration in the means of O2sat, PaO2, paCO2, pH and RR between both groups (P > 0.05). However, after intervention, the means of O2sat, PaO2 in the case group were significantly higher than the control group (P < 0.0001). Conversely, the means of the PaCO2 and RR in the control group were significantly higher than the case group (P < 0.0001). Table 3 displays the comparison of the median of nocturnal enuresis frequency between the groups before and after intervention. Before intervention, the median of nocturnal enuresis frequency was similar in both groups (P > 0.001). After intervention, the median of nocturnal enuresis frequency in the case group was significantly lower than the control group (P < 0.001). Table 3. Comparison of the Nocturnal Enuresis Frequency between the Groups. Before After P value a Case Group b 6 (4.25 – 8) 1 (0 – 2) < 0.0001 Control Group b 6 (4.25 – 7.75) 5.5 (4 – 8.75) 0.83 P value c 0.7 < 0.0001 a P-values were calculated by the Wilcoxon Signed Rank tests, b Values are represented as median (IQR). c P-values were calculated by the Mann-Whitney. BODY.5. DISCUSSION: This study aimed to consider the effects of breathing exercises on the nocturnal enuresis in the children with the sleep-disordered breathing. Normally, in each breath, 500 ml of air enter the lungs, and then it goes out of the lungs after the exchange of oxygen and carbon dioxide. With deep breathing, the amount of air can be increased largely. These breathing exercises bring the air volume to 2500 ml in each breath. Therefore, by breathing exercises, more air will enter the lungs, arterial blood oxygen saturation will increase, and respiratory proper functioning will be possible, which promotes deep breathing improvement (11, 25, 26). Breathing exercises will also prevent the accumulation of secretions and atelectasis, strengthen the respiratory muscles, coordinate aspiration muscles and correct the breathing patterns. It also decreases the asthmatic attacks and eliminates the dyspnea (14). On the contrary, in children with the oral breathing during sleep, the upper airways endure vibration due to airflow, and breathing becomes difficult due to weakening of the respiratory muscles. In addition, the chronic superficial breathing causes the weakening of the inter-rib muscles and diaphragm muscle (17, 18). Many studies show that the removal of airways blockade even in case of surgery, reduces the frequency of nocturnal enuresis in children with the breathing disorders (16). There are some drugs and various behavioral to treat the nocturnal enuresis, but none of them is crucial, and each has side effects. On the other hand, the treatment of nocturnal enuresis with respect to the associated disorders, can be more effective/with less complications (2, 27, 28). The results of this study showed that in children with the nocturnal enuresis, who had the blockade of airways/abnormal breathing pattern; the breathing exercises can be used as a simple method with low expenses and more efficient than other approaches. The findings of this study also showed that there was no significant difference in the demographic characteristics between the two groups (Table 1). In addition, the findings of this study revealed that there was a significant difference in the mean frequency of nocturnal enuresis between the case and the control groups after the intervention (Table 3). These findings demonstrate that the frequency of breathing exercises reduce the nocturnal enuresis in the children with the sleep-disordered breathing. There are many studies concerning the relationship between the nocturnal enuresis and nocturnal snore/airways blockade ( 16 , 21 , 22 , 29 ). The nocturnal enuresis is more common in children with the oral breathing. It appears that the relative hypoxia in these children causes low-pressure oxygen in the distal tubes of kidney, unresponsiveness to the anti-diuretic hormone, and increased production of urine temporarily, particularly during the sleep. Both of these factors can increase the volume of urine, and consequently make the urine control more difficult and finally lead to the nocturnal enuresis ( 3 , 7 ). As far as we are concerned, there is no internal research concerning the impact of breathing exercises on the nocturnal enuresis in children. Nevertheless, Crouch et al. have shown that in subjects with the breathing disorders, the pulmonary rehabilitation programs including breathing exercises, endurance activities, upper and lower extremities exercises like purse lip breathing and diaphragmatic breathing, improve ventilation and in turn, have caused the adequate exchange of air pattern ( 19 ). Ghahri Sarabi et al. have also shown that in patients with the breathing disorders, breathing exercises increase the lung functional indices ( 27 ). In addition, another study has shown that in patients with the respiratory chronic diseases, pulmonary rehabilitation and breathing exercises increase the volume of oxygen consumption ( 20 ). The respiratory disorders cause ineffective breathing habits and shallow breathing. This chronic shallow breathing leads to the weakening of the diaphragm muscle and inter-costal muscles ( 8 , 19 ). In conclusion; these findings suggest that the breathing exercises may reduce the nocturnal enuresis in the children with the sleep-disordered breathing. The clinical implications of these findings should be verified in the future longitudinal studies.

TITLE: The evaluation of effects two different doses of hydrocortisone on the intensity of perioperative shivering in elective surgery under spinal anesthesia: A double-blind randomized controlled trial study ABSTRACT.BACKGROUND:: Post- and intra-operative shivering is one of the most complications of spinal anesthesia so recommend a suitable drug with at least complications for prevention and control of postoperative shivering. This current study aimed to compare the preventive effect of hydrocortisone on intra- and post-operative shivering in patients undergoing surgery with spinal anesthesia. ABSTRACT.MATERIALS AND METHODS:: In a clinical trial study, ninety patients who candidate for surgery with spinal anesthesia were selected and randomly divided into three groups. The first and second groups were received 1 mg/kg and 2 mg/kg hydrocortisone, respectively, and the third group was received normal saline, and postoperative shivering was compared between the three groups. ABSTRACT.RESULTS:: The investigation of the incidence of inter- and post-operative shivering in patients in the three groups revealed that within the study period, 31 patients suffered from shivering among which 9, 5, and 17 cases were in 1 mg/kg hydrocortisone group, 2 mg/kg hydrocortisone group, and placebo group, respectively, and according to the Chi-square test, the difference among the three groups was significant (P = 0.004). ABSTRACT.CONCLUSION:: According to the obtained results, the overall conclusion of the study is that using hydrocortisone at least with the dose of 1 mg/kg as a preventive drug reduced the incidence of intra- and post-operative shivering with spinal anesthesia. BODY.INTRODUCTION: The reactions of the immune system to temperature loss include activities of skin vasomotor, thermogenesis, sweating, and shivering, which is indeed the last immune mechanism, is produced in associated with muscular activities with a frequency of 4–8 Hz, and reinforces the temperature metabolism to 600% compared to the base level.[123456] Some processes can lead to central hypothermia. General and regional anesthesia are the same and in both the distribution of heat is from the center to the initial process environment that in spinal and epidural, the process does not reach to the balance because the environmental vasoconstriction is lost and shivering and low temperature are produced by a small volume of muscles above the block level.[78] Shivering occurs in 50% of patients with the core temperature of 35.5°C and in 90% of cases with the core temperature of 34.5°C.[9] Postoperative shivering is a common complication of spinal anesthesia which has different incidence in various studies and has been expressed up to 40–56.7% depending on the type and consumed drug.[1011] To treatment and prevent postoperative shivering, different drugs can be used that among from opioid drugs, pethidine, fentanyl, and tramadol and out of nonopioid drugs, dexamethasone, ondansetron, clonidine, pentazocine, ketamine, and magnesium sulfate can be mentioned.[7] Hydrocortisone is corticosteroids similar to the natural hormone produced by the adrenal gland that causes to remove inflammation (sweating, heat, redness, and pain) and its intravenous injection controls postoperative shivering with general anesthesia. The mechanism of hydrocortisone effect is applied through anti-inflammatory mechanism, core temperature gradient, and skin.[11] Since it is tried to recommend a suitable drug with at least side complications for the prevention and control of postoperative shivering, most conducted studies emphasize on the treatment of shivering, and prophylaxis has been studied less; on the other hand, until now, such study has not been conducted on people with spinal anesthesia and given the prevalence and increase of surgeries with spinal anesthesia, the current study aimed to compare the preventive effect of hydrocortisone on the incidence and intensity of shivering in patients undergoing surgery with spinal anesthesia. BODY.MATERIALS AND METHODS: This is a randomized, double-blind clinical trial study conducted in an educational hospital (Kashani Hospital) of Isfahan University of Medical Sciences in Isfahan, Iran on 2013–2014. Patients were selected from those who were referred for elective orthopedic operation. Inclusion criteria were the age range of 18–60-year-old, patients undergoing lower extremity orthopedic operation with spinal anesthesia (knee arthroplasty, hip arthroplasty, femoral fracture...) American Society of Anesthesiologists physical status I–II,[2] no having hypothyroidism, hyperthyroidism, cardiopulmonary diseases, psychological disorders, no alcohol and drugs, lack of using vasodilator or drugs affecting the body's temperature settings, and patient's consent to participate in the study. It was also decided that patients requiring blood transfusions during operation, with the base temperature more than 38°C and <36°C or change in the techniques of anesthesia were excluded from the study. We assumed the incidence of shivering in the case and control group 10% and 40%, respectively, due to previous studies[56] with respect to α =0.05 and β =0.2. We calculated 29 patients in each group as the sample size. The method was in a way that after the approval of proposal and adoption of consent of Medical Ethics Committee, ninety patients with inclusion criteria were selected and divided into three 30-case equal groups by using random allocation software. Exactly, 1 mg/kg hydrocortisone + 2 cc normal saline, 2 mg/kg hydrocortisone + 2 cc normal saline, and the same normal saline (2 cc) were injected to patients in M, P, and C (control) groups, respectively. The volume of each syringe was maximally 2 cc. The syringe was similar with respect to the shape and color. The study drugs were administered 10 min before spinal anesthesia. Fluid therapy and anesthesia method were the same in all three groups. All patients were NPO for 8 h and during this time, the fluid therapy was done according to 4-2-1 formula of serum (4 ml/kg/h for the first 10 kg of body weight, 2 ml/kg/h for the second 10 kg of body weight, and 1 ml/kg/h for the third 10 kg of body weight) and then intravenous midazolam 0.05 mg/kg was injected to all patients 5 min before the onset of anesthesia. Before the onset of anesthesia, 10 cc/kg of Ringer Lactate was injected to all patients within 30 min, and patients were routinely monitored including pulse oximetry, automatic control blood pressure, central and peripheral temperature, and 3-lead electrocardiogram. Oxygen was given to the patients via a face mask as 5 l/min until the end of the operation. Spinal anesthesia was performed at L4–L5 or L3–L4 of the spinal needle of 24-gauge, midline, and at sitting position. After leaving transparent and cerebrospinal fluid, 2.5 cc bupivacaine of 0.5% was injected within 10 s for the anesthesia. Then, the patient was immediately placed at supine position and the patient's head was put 15–20°C above the horizon and no analgesic drug was injected into the patient. The level of sensory block was determined using bilateral pin-prick test in midclavicular line. When the level of block reached to the desired level, the lower limb operation was started. The temperature of the operating room had been maintained at 21–22° and nonoperative parts were covered by a layer of cloth. After the operation, the whole parts of the body were covered by a layer of cotton blanket and then, the patients were transferred to the recovery room. The temperature of recovery room was similar for all patients, and no heating device was individually used for patients. The baseline level of the central and peripheral temperatures was measured. Moreover, immediately before anesthesia (on operating table), immediately after the anesthesia, 5 min after anesthesia, before and after drug injection, every 10 min during the operation, at the onset of entrance to the recovery room, and after recovery at 10, 20, 30, 40, 50, and 60 min, central and peripheral temperatures were measured by tympanic thermometer (OMRON Medizintechnik GmbH, Mannheim, Germany) and an axillary thermometer (BMEcenter.ir, Iran). If the peripheral temperature was below 36°C, hypothermia would be considered. In hypothermic patients, if they had shivering with grade = 4. Meperidine 0.4 mg/kg was administered. To ensure that the study was double-blinded (both patients and physician who recorded the data), drugs were encoded by an operating room staff. All patients received postoperative routine nursing care and in all cases, basic rate of parameters including blood pressure, respiratory rate, heart rate, shivering score (0, no shivering; 1, piloerection or peripheral vasoconstriction but no visible shivering; 2, muscular activity in only one muscle group; 3, muscular activity in more than one muscle group but not generalized; and 4, shivering involving the whole body), pain score, drowsiness score, and vomiting was recorded. The incidence and intensity of shivering was evaluated during intra- and post-operative periods. Hypotension (low systolic pressure below 100 mmHg or 25% reduction in systolic blood pressure compared to patients' baseline pressure) was treated by intravenous Ringer lactate serum and 5 mg intravenous ephedrine. If the heart rate drops below 55 rates, the patients would receive 0.01 mg intravenous atropine per kg of body weight and if necessary, it would be repeated up to a maximum dose of 0.04 mg per kg of body weight. You should declare that as the ephedrine and atropine could alter the body temperature these nausea and vomiting were treated by 10 mg intravenous metoclopramide. All the received drugs, the number of frequency, and their dose were recorded. If patients had shivering Grade 4, meperidine 0.4 mg/kg was administered. The degree of sedation was evaluated as 1, fully awake and oriented; 2, drowsy; 3, eyes closed but rousable to command; 4, eyes closed but rousable to mild physical stimulation; and 5, eyes closed but unrousable to mild physical stimulation. The study data were entered into the computer after collecting and were analyzed using SPSS software version 20. Chi-square test (for comparison of qualitative data between groups), one-way analysis of variance (ANOVA) (for comparison of quantitative date between groups), and repeated measure ANOVA (for comparison of trend of quantities date between groups) with repeated observations, and median test were run to analyze the data. BODY.RESULTS: In this study, ninety patients undergoing surgery with spinal anesthesia were investigated and assessed that during the study, no patient excluded from the study due to the incidence of unwanted complications such as hemodynamic and in the anesthesia method and all ninety patients were present in the study until the end of the study. In Table 1, the distribution of demographic and general variables of patients in the three groups has been shown. Table 1 Distribution of demographic and general variables in three study groups The investigation of the incidence of shivering in patients in the three groups revealed that within the study period, 31 patients suffered from shivering among which 9, 5, and 17 cases were in the 1 mg/kg hydrocortisone group, 2 mg/kg hydrocortisone group, and placebo group, respectively, and according to the Chi-square test, the difference among the three groups was significant (P = 0.004). Moreover, the intensity of shivering had no significant difference among the three groups (P = 0.046). The distribution of the frequency of the incidence and intensity of shivering in the three groups are given in Table 2. Comparing the incidence and intensity of shivering in binary groups indicated that the two groups of hydrocortisone of 1 mg/kg and 2 mg/kg had no significant difference in terms of the incidence and intensity of shivering (P = 0.23 and P = 0.2, respectively), while the incidence and intensity of shivering had a significant difference between the two groups of hydrocortisone of 1 mg/kg and placebo group (P = 0.039 and P = 0.018, respectively). Moreover, the incidence and intensity of shivering had a significant difference between the two groups of hydrocortisone of 2 mg/kg and placebo group (P = 0.0001 and P = 0.001, respectively). The score of sedation in patients in the three groups had no significant difference from the onset of the induction of anesthesia to 60 min of recovery in none of the studied periods. Within the study period, no patient suffered from drug allergy, whereas out of hydrocortisone group 2 mg/kg, one patient suffered from bradycardia and one patient suffered from hypotension. One of the hydrocortisone 1 mg/kg group and 1 case of hydrocortisone 2 mg/kg group suffered from postoperative nausea and vomiting, while no significant difference was observed among the three groups (P = 0.6). Table 2 Distribution of the frequency of the incidence and intensity of shivering in the three groups Figures 1 and 2 shown the mean central and peripheral temperatures in the three studied groups from the onset of the operation until 60 min of recovery. According to the one-way ANOVA test, the mean central and peripheral temperatures had no significant difference between the three groups in any of the study periods. Furthermore, repeated measure ANOVA indicated that the process of changes in the central and peripheral temperatures had no significant difference among the three groups (P = 0.64 and P = 0.31, respectively). The lowest core and peripheral temperatures were 35.75°C and 35.70°C, respectively, which occurred 90 min after spinal anesthesia. Figures 3 and 4 shown the Mean blood pressure from 0 to 60 min recovery in the three groups (P = 0.82) and Mean heart rate from 0 to 60 min recovery in the three groups (P = 0.77). Repeated measure ANOVA indicated that had no significant difference among the three groups (P = 0.82 and P = 0.77). Figure 1Mean central temperature from 0 to 60 min recovery in the three groups (P = 0.64) Figure 2Mean peripheral temperature from 0 to 60 min recovery in the three groups (P = 0.31) Figure 3Mean blood pressure from 0 to 60 min recovery in the three groups (P = 0.82) Figure 4Mean heart rate from 0 to 60 min recovery in the three groups (P = 0.77) BODY.DISCUSSION: Shivering is the involuntary movements of one or several muscle groups and generally occurs in the initial phase of recovery after anesthesia. The incidence of this condition varies between 6.3% and 66% depending on the type and duration of operation, anesthesia technique, and patient's age and gender[12345678910] and has minimum incidence to 40% in patients operation with spinal anesthesia.[1011] With regard to the incidence and clinical significance, shivering has been known as the sixth major problem among from 33 clinical cases with the low rate of morbidity in clinical anesthesiology.[12] Of postoperative complications, the increase in oxygen consumption at the rate of 200–500%, production of CO2, heart rate and stroke volume, and blood pressure can be pointed out that their suppression reduces the metabolic need and myocardial function.[131415161718] Other complications include the increase of intraocular pressure and intracranial pressure, creation of problem in monitoring the patient, and interference with the surgical cares, especially in cases requiring immobilization, for example, secular or nerve anastomosis.[141516] According to the results of our study, three studied groups had no significant difference in terms of demographic variables and operation features such as duration of operation and anesthesia and sensory block level and no confounding effect of the above-mentioned factors was observed on the incidence and intensity of shivering and therefore it may pertain to the fact that the difference in the incidence of postoperative shivering is related to the type and rate of preventive consuming drug. According to the results of our study, the incidence of shivering in the group receiving hydrocortisone including a dose of 1 mg/kg or 2 mg/kg was considerably and significantly less than the control group. On the other hand, patients receiving hydrocortisone had lower shivering; however, no significant difference was observed between the two doses of the drug. A number of pharmaceutical interventions for the treatment and prophylaxis of shivering including clonidine, ketamine, doxapram, tramadol, pethidine, and other opioids have so far been examined (14, 17, and 18). In the study by Pawar et al., in 2011, the effectiveness of hydrocortisone for the prevention of shivering was approved so that the incidence of shivering was 32% in patients receiving hydrocortisone and 82% in the control group.[18] In their study, Yousef and Johnson showed that using hydrocortisone as a preventive drug reduced the incidence of hypothermia; consequently, it reduced the incidence of shivering in patients undergoing an operation.[19] Therefore, according to the obtained results, the overall conclusion of the study is that using hydrocortisone at least with the dose of 1 mg/kg as a preventive drug reduced the incidence of shivering with spinal anesthesia. However, because limitations of our study such as small sample size, to highly ensure the effect of dose-response of the drug, further studies are recommended. BODY.CONCLUSION: According to this study using hydrocortisone at least with the dose of 1 mg/kg as a preventive drug reduced the incidence of intra- and post-operative shivering with spinal anesthesia. BODY.CONCLUSION.FINANCIAL SUPPORT AND SPONSORSHIP: Anesthesiology and Critical Care Research Center, Isfahan University of Medical Sciences, Isfahan, Iran. BODY.CONCLUSION.CONFLICTS OF INTEREST: The authors have no conflicts of interest. BODY.AUTHORS’ CONTRIBUTION: MRS has planned the study and finalized it; FK, AH, MN, HS, and MRS did the statistical analysis and prepared the first version of the manuscript for publish. All authors read and approved the final manuscript.

TITLE: Management of osteoporosis and menopausal symptoms: focus on bazedoxifene/conjugated estrogen combination ABSTRACT: Loss of estrogen production in women during menopause results in a state of estrogen deficiency which has been associated with multiple problems, including vasomotor symptoms, symptoms of vulvovaginal atrophy, bone loss, and difficulties with sleep, mood, memory, and sexual activity. The only treatment option currently available to address multiple postmenopausal symptoms in women with an intact uterus is estrogen/progestin-containing hormone therapy (HT). Concerns surrounding side effects and published data regarding the association of HT with the increased risk for breast cancer have induced a decrease in the number of women seeking, initiating, and continuing this type of therapy. A combination containing bazedoxifene and conjugated estrogens (BZA/CE) maintains the established benefits of estrogen therapy for treatment of postmenopausal vasomotor symptoms, vulvovaginal atrophy, and osteoporosis, while certain estrogenic effects, such as stimulation of the uterus and breast, are antagonized without the side effects associated with HT. BZA/CE has been evaluated in a series of multicenter, randomized, double-blind, placebo-controlled, and active-controlled Phase III trials known as the Selective estrogens, Menopause, And Response to Therapy (SMART) trials. BZA/CE demonstrated clinically meaningful improvements in vasomotor symptoms, vulvovaginal atrophy, and a protective effect on the skeleton. These clinical benefits were associated with an acceptable safety profile and an improved tolerability compared with HT. BZA/CE showed a favorable safety profile on the breast, endometrium, and ovaries. The incidence of venous thromboembolism was low and the risk does not appear to be any greater than for CE alone or BZA alone or greater than HT. The incidence of coronary heart disease and cerebrovascular accidents were similar to placebo. The overall incidence of cancer (including breast cancer) was low and similar to placebo. The SMART trials demonstrate that BZA/CE is an alternative option for treating non-hysterectomized, symptomatic, postmenopausal women. BODY.INTRODUCTION: Loss of ovarian estrogen production in women during menopause results in a state of estrogen deficiency1 which has been associated with multiple problems, including vasomotor symptoms, symptoms of vulvovaginal atrophy, and difficulties with sleep, mood, memory, and sexual activity.2 During menopause, these symptoms commonly overlap, presenting as a continuum, and may have a negative impact on a woman's quality of life.3 Menopausal symptoms have also been negatively associated with the ability to work.4 Estrogen deficiency has further been associated with loss of bone mass, often leading to osteoporosis, which is associated with an increased occurrence of skeletal fractures.2 Women who sustain osteoporotic fractures also face a negative impact on their quality of life, often affecting their ability to function and putting them at an increased risk of morbidity and mortality.2,5,6 A variety of therapeutic options are available for the individual symptoms associated with menopause. Estrogen/progestin-containing hormone therapy (HT) is the only currently approved treatment for women with a uterus that holistically addresses the postmenopausal problems of vasomotor symptoms and vulvovaginal atrophy, while maintaining bone mass.3 HT has pharmacologic drawbacks, including vaginal bleeding and breast pain/tenderness, which are the most common reasons for discontinuation of this therapy.7–9 Vaginal bleeding leads to discontinuation of therapy in up to one-third of women and rates of discontinuation can be as high as 68% within the first year of treatment.10 Rates of discontinuation for breast tenderness/pain have been reported to be as high as 40% in some clinical trials.11,12 Progestin-induced irregular vaginal bleeding associated with HT creates distress and often requires additional interventions (ie, transvaginal ultrasonography, endometrial biopsies, and hysteroscopies).13 Increases in breast density associated with progestin-containing HT may lead to mammographic recalls because of difficulty in reading mammogram results. Mammographic recalls also increase anxiety in women, contributing to the decision to discontinue HT.14,15 Women's concerns about the available therapeutic options for postmenopausal symptoms have resulted in a substantial population who are currently untreated.3 The population of postmenopausal women will grow, given the continual increase in the numbers of women approaching menopause and the extended life expectancy of postmenopausal women.1 Thus, for postmenopausal women with a uterus, new therapeutic alternatives with an improved tolerability and safety profile compared with traditional HT are needed to help alleviate vasomotor symptoms and vulvovaginal atrophy symptoms, and to prevent osteoporosis. BZA/CE combines bazedoxifene (BZA), a selective estrogen receptor modulator, with conjugated estrogens (CE), and represents a new treatment option in the management of menopausal health for women with a uterus. BZA/CE maintains the established benefits of estrogen therapy for menopausal symptoms of vasomotor symptoms, vulvovaginal atrophy, and prevention of osteoporosis, while preventing the stimulatory estrogenic effects on the uterus and estrogenic/progestogenic effects on breast tissue. The rationale for development of BZA/CE was that BZA, acting as an estrogen receptor antagonist in uterine tissue, would inhibit the proliferative effects of estrogen on the endometrium in a manner mechanistically distinct from progestins, and therefore reduce the incidence of irregular uterine bleeding. BZA acts as an estrogen receptor antagonist in breast tissue, so BZA/CE is posited not to induce breast pain or changes in breast density. BZA/CE does not cause morphologic changes in the mammary gland, and it is hypothesized that this is the pharmacologic explanation for the lack of changes in breast pain and density. In vitro and vivo models demonstrate that BZA also inhibits the proliferative stimulatory action of estrogens on breast cancer cells.16 The objective of this review is to present key efficacy and safety findings from the Selective estrogens, Menopause, And Response to Therapy (SMART) trials.17–20 BODY.CLINICAL STUDIES WITH BZA/CE: BZA/CE has been evaluated in a series of multicenter, randomized, double-blind, placebo-controlled, and active-controlled Phase III investigations known as the SMART trials (Table 1).17–20 The SMART-1 trial (n = 3,397) was conducted at 94 sites in the United States, Europe, and Brazil and enrolled generally healthy, postmenopausal women aged 40–75 years.17 Eligible subjects had to have an intact uterus, a body mass index (BMI) ≤32.2 kg/m2, and normal endometrial biopsy results at screening. SMART-1 evaluated the efficacy of multiple doses of BZA/CE (combinations of BZA 10 mg, 20 mg, and 40 mg with either CE 0.45 mg or CE 0.625 mg) with regard to uterine protection, vasomotor symptoms, vulvovaginal atrophy, and prevention of osteoporosis.17 The one-year interim results from SMART-1 demonstrated that BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg have a low (<1%) incidence of endometrial hyperplasia, while reducing the number and severity of hot flushes, improving symptoms of vulvovaginal atrophy, and preventing bone loss. Therefore, BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg were selected for further evaluation in other SMART trials.17 The SMART-2 trial (n = 318) was conducted at 43 sites in the United States and enrolled healthy, postmenopausal women aged 40–65 years with an intact uterus and a BMI ≤ 34.0 kg/m2. Eligible subjects had to have seven or more moderate to severe hot flushes daily (or at least 50 per week) at screening and be seeking treatment for hot flushes. This study evaluated the effects of BZA 20 mg/CE 0.45 mg and 0.625 mg on vasomotor symptoms compared with the effects of placebo over 12 weeks of treatment. The primary endpoint was change from baseline in the frequency and severity of hot flushes. Secondary endpoints included effects on sleep, quality of life, and satisfaction with treatment.18 The SMART-3 trial (n = 652) was conducted at 66 sites in the United States and enrolled generally healthy, postmenopausal women aged 40–65 years with an intact uterus and a BMI ≤ 34.0 kg/m2. All women had to have a vaginal cytologic smear showing no more than 5% superficial cells, a vaginal pH > 5, and at least one moderate to severe vulvovaginal atrophy symptom at screening. This study examined the effects of BZA 20 mg/CE 0.45 mg and 0.625 mg compared with those of BZA 20 mg alone and placebo on measures of vulvovaginal atrophy over 12 weeks of treatment.19 The SMART-5 trial (n = 1,886) was conducted at 166 sites in the United States, Europe, Latin America, Australia, and New Zealand, and enrolled generally healthy, postmenopausal women aged 40–75 years. Eligible subjects had to have an intact uterus, a BMI ≤ 34 kg/m2, and acceptable endometrial biopsy results at screening. SMART-5 evaluated the efficacy of BZA/CE on uterine protection, prevention of osteoporosis, and effect on breast density.20 This study evaluated the effects of BZA 20 mg/CE 0.45 and 0.625 mg and included CE/medroxyprogesterone acetate (MPA, (Prempro®, Pfizer Pharmaceuticals Inc, New York, NY, USA), BZA, and placebo as comparators. The results of the one-year SMART-4 study (n = 1,061) are not discussed in this review because a different formulation of BZA/CE was evaluated. Regardless, the results of the SMART-4 study were similar to those of the SMART-1 study.17 BODY.EFFICACY ON HOT FLUSHES, HEALTH RELATED-QUALITY OF LIFE, AND SLEEP: Two clinical trials (SMART-1 and SMART-2) evaluated the efficacy of BZA/CE for the treatment of moderate to severe vasomotor symptoms. In these studies, BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 were associated with clinically meaningful improvements in vasomotor symptoms as compared with placebo, and the efficacy was comparable with that of available hormonal options (HT and estrogen therapy).17,18 In SMART-2, at week 4, BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg were associated with a significant decrease (P < 0.001) in the adjusted mean daily number of moderate to severe hot flushes as compared with placebo. A clinically meaningful reduction of 5–6 hot flushes per day was observed in subjects treated with BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg, respectively, compared with a reduction of three hot flushes in the placebo group. At week 12, BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg were associated with a decrease (P < 0.001) in the adjusted mean daily number of moderate to severe hot flushes, reaching a 74% and a 80% reduction, respectively, representing a clinically meaningful reduction of approximately 7–8 hot flushes per day, compared with a 51% reduction (representing a reduction of approximately five hot flushes per day) in the placebo group (Figure 1).18 In addition to significant reductions in the number of moderate to severe hot flushes experienced per day, both BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg dose strengths demonstrated clinically meaningful reductions in the severity of moderate to severe hot flushes compared with subjects who received placebo at week 4 (26% and 29% reduction, respectively) and week 12 (39% to 55% reduction, respectively, P < 0.001, Figure 2).18 Similar results were observed in SMART-1, with evidence of efficacy of BZA/CE for the treatment of vasomotor symptoms present through 2 years of therapy.17 BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 was associated with clinically meaningful improvements in vasomotor symptoms. Comparison with historical data reveals that both the BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg dose strengths have an overall comparable efficacy with that of estrogen progestin-containing HT compounds in the relief of moderate to severe hot flushes associated with menopause.21 Both BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg treatment groups demonstrated efficacy for both the daily number and severity of moderate to severe hot flushes, regardless of racial characteristics, BMI, and years since menopause of the populations evaluated.18 Health-related quality of life, sleep parameters, and patient satisfaction tools were also used to assess patient response to therapy in the SMART-2 study. Clinically significant improvements were reported with BZA 20 mg/CE 0.45 and 0.625 mg at 12 weeks compared with placebo in the vasomotor function domain of the Menopause-Specific Quality of Life questionnaire and significantly greater satisfaction in the ability to control hot flushes during the day and night according to the Menopause Symptoms Treatment Satisfaction Questionnaire.22 A range of components of the Medical Outcomes Study sleep scale also showed significant improvements with BZA/CE versus placebo, including significant decreases in sleep disturbance and significant increases in sleep adequacy.22 Additionally, BZA/CE improved both indices (sleep prob­lem index 1 and 2) included in the Medical Outcomes Study sleep scale (Figure 3). Statistical mediation modeling dem­onstrated that BZA/CE affected sleep directly in populations of women with severe vasomotor symptoms, while in less symptomatic women its effect was indirect via improvement in hot flushes.22 In summary, BZA/CE was associated with a clinically meaningful reduction in vasomotor symptoms, comparable with that observed for HT; moreover, this efficacy was correlated with improvements in sleep and menopause-related quality of life. BODY.EFFECTS ON VULVOVAGINAL ATROPHY: Two studies evaluated the efficacy of BZA/CE on vulvovaginal atrophy, ie, SMART-1 and SMART-3.17,19 In the SMART-3 trial, the efficacy of BZA 20 mg/CE 0.45 and 0.625 mg on vulvovaginal atrophy was evaluated in subjects who had no more than 5% superficial cells at screening on the vaginal smear, a vaginal pH < 5, and presented with a moderate to severe symptom associated with vulvovaginal atrophy.19 BZA 20 mg/CE 0.45 or 0.625 mg showed a significantly greater increase from baseline in the mean proportion of superficial cells compared with placebo (P < 0.001). Both BZA/CE groups also showed a significantly greater reduction from baseline in the mean proportion of parabasal cells (P < 0.001) and a significantly greater increase from baseline in the mean proportion of intermediate cells (P < 0.05) compared with placebo at weeks 4 and 12. Additionally, treatment with BZA 20 mg/CE 0.625 mg resulted in a significantly greater decrease from baseline in vaginal pH (P < 0.001) and a greater improvement in the women's most bothersome vulvovaginal atrophy symptoms (P < 0.05) compared with placebo (Figure 4).19 In SMART-1, BZA/CE showed a similar effect on the vaginal epithelium, inducing a greater increase from baseline in the mean proportion of superficial cells compared with placebo (P < 0.001), a greater increase in the mean proportion of intermediate cells (P < 0.001), and a greater decrease in the mean proportion of parabasal cells from baseline compared with placebo (P < 0.001). This maturation of the vaginal epithelium was associated with a statistically significant reduction in the incidence of dyspareunia relative to placebo during weeks 9–12 of therapy (P < 0.001) and in ease of lubrication score from baseline compared with placebo (P < 0.05) on the Arizona Sexual Experiences scale.17,19 Additionally, in SMART-3 (subjects with vulvovaginal atrophy), the Menopause-Specific Quality of Life questionnaire results at week 12 showed significant improvements in sexual function and total scores with both BZA/CE doses versus placebo or BZA 20 mg (P < 0.001). The Menopause Symptoms Treatment Satisfaction Questionnaire results showed that BZA/CE-treated subjects reported significantly greater overall satisfaction with treatment.19 BODY.BONE EFFECTS: Two clinical trials (SMART-1 and SMART-5) evaluated the safety and efficacy of BZA/CE for the prevention of osteoporosis.17,20 Among subjects ≤5 years since menopause (substudy II of SMART-1 and SMART-5), significant (P < 0.001) increases from baseline to month 12 in lumbar spine bone mineral density (BMD) were demonstrated for both the BZA 20 mg/CE 0.45 mg (1.05% and 0.24% in SMART-1 and SMART-5, respectively) and BZA 20 mg/CE 0.625 mg (1.05% and 0.60%, in SMART-1 and SMART-5, respectively) treatment groups compared with placebo (−1.81% and −1.28%, in SMART-1 and SMART-5, respectively).17,20 In addition, in SMART-1, a statistically significant increase in lumbar spine BMD from baseline to month 24 was observed for both the BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg treatment groups compared with a decrease in lumbar spine BMD in the placebo group (P < 0.001). Raloxifene 60 mg was included as an active comparator in SMART-1; among subjects ≤5 years since menopause, both the BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg dose strengths demonstrated a statistically significant greater percentage increase in lumbar spine BMD from baseline to months 12 (P < 0.001) and 24 (P = 0.002 and P = 0.001, respectively) compared with raloxifene 60 mg (Figure 5).17 In SMART-5, BZA 20 mg and CE 0.45 mg/MPA 1.5 mg were included as active comparators. BZA/CE (both doses) showed similar efficacy to CE/MPA and a superior bone effect as compared with BZA 20 mg.20 SMART-1 and SMART-5 demonstrated that treatment with BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg effectively prevented loss of total hip BMD, having a comparable effect to that of raloxifene 60 mg and BZA 20 mg at all time points evaluated. In addition, BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg demonstrated a comparable effect to CE 0.45 mg/MPA 1.5 mg in terms of effects on BMD at the total hip.17,20 The results from subpopulation analyses based on the integrated data from SMART-1 and SMART-5 demonstrate that BZA/CE therapy was efficacious regardless of age groups (<60 years versus ≥60 years), BMI, race, and geographic region in improving lumbar spine and total hip BMD.17,20 To evaluate further the effect of treatment on bone metabolism, serum samples for determination of osteocalcin, procollagen type 1 N-propeptide (P1NP), and C-telopeptide were obtained in SMART-1 and SMART-5.17,20 The observed decreases in bone turnover markers were consistent with the observed improvements in BMD. Both the BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg treatment groups demonstrated significant decreases from baseline in serum concentrations of C-telopeptide and osteocalcin compared with placebo (P < 0.001) in SMART-1 and SMART-5. In addition, concentrations of P1NP at months 6 and 12 were significantly decreased (P < 0.001) by both doses compared with placebo in SMART-5.17,20 A significantly larger decrease (P < 0.001) in serum concentrations of C-telopeptide and osteocalcin was observed for both the BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg treatment groups compared with the raloxifene 60 mg treatment group in SMART-1.17 Similarly, in SMART-5, both the BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg treatment groups demonstrated greater decreases in serum osteocalcin and serum C- telopeptide and significant decreases in P1NP compared with the BZA 20 mg treatment group. Moreover, in SMART-5, both the BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg treatment groups demonstrated decreases in P1NP and serum osteocalcin that were comparable with the CE 0.45 mg/MPA 1.5 mg treatment group.20 Studies conducted with BZA alone provide important supportive evidence for the long-term efficacy (7-year exposure) of BZA in postmenopausal subjects. Treatment with BZA 20 and BZA 40 mg for 3 years resulted in a statistically significant reduction (P < 0.05) in the incidence of new vertebral fractures compared with placebo which was similar to that observed with raloxifene 60 mg. This positive effect in reduction of new vertebral fractures was observed through 5 and 7 years of treatment.23 Overall, the skeletal effects (changes in lumbar spine and hip BMD and bone turnover markers) observed with BZA/CE therapy were clinically meaningful and greater than for raloxifene and BZA monotherapy and comparable with CE 0.45 mg/MPA 1.5 mg. The increases in BMD and decrease in bone turnover markers observed with BZA/CE as compared with placebo have been associated with a reduction in fracture risk with antiresorptive agents as well as with BZA treatment. BODY.SAFETY.ENDOMETRIAL SAFETY: Protection of the endometrium is of major clinical importance for non-hysterectomized postmenopausal women who receive a therapy containing estrogens. Therefore, the tissue selective activity of the selective estrogen receptor modulator component of BZA/CE is key to endometrial protection.16 The incidence of endometrial hyperplasia with BZA/CE was evaluated in SMART-1 and SMART-5. These studies were of sufficient duration to provide evidence that BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg provides appropriate endometrial protection. Both doses of BZA/CE demonstrated an incidence of endometrial hyperplasia <1%, which was comparable with placebo.17,24 Data obtained using transvaginal ultrasound have also shown neutral effects of BZA/CE on the endometrium. BZA/CE were associated with minimal increases in endometrial thickness from baseline (<1 mm) which were similar to placebo.20 More subjects treated with CE/MPA in the SMART-5 trial had an increase in endometrial thickness of >5 mm compared with BZA/CE or placebo.20 The effects of BZA/CE on endometrial bleeding have also been evaluated in the SMART trials.20,25 In SMART-1 and SMART-5, BZA 20 mg/CE 0.45 and 0.625 mg were associated with high rates of cumulative amenorrhea, similar to placebo. The rates of cumulative amenorrhea over one year were >83%, >87%, and >85% for BZA 20 mg/CE 0.45 mg, BZA 20 mg/CE 0.625 mg, and placebo, respectively, in SMART-1.25 Higher rates of cumulative amenorrhea were observed with BZA 20 mg/CE 0.45 and 0.625 mg (>87% and >84%, respectively) and placebo (>83%) compared with CE/MPA (>54%) in SMART-5 (Figure 6).20 BODY.SAFETY.BREAST SAFETY: The effect of BZA/CE regimens on the incidence of breast pain/tenderness was evaluated in SMART-1, SMART-2, and SMART-5.17,18,20 In these studies, breast pain/tenderness was measured by daily subject diaries. Both the BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg dose strengths demonstrated an incidence of breast pain/tenderness that was not significantly different compared with placebo, raloxifene 60 mg, and BZA 20 mg.17 In SMART-5, the incidence of breast tenderness in subjects treated with BZA/CE was significantly lower than that in subjects treated with CE 0.45 mg/MPA 1.5 mg (P < 0.001, Figure 7).20 These results are consistent with the relative incidences of adverse events related to breast pain/tenderness and discontinuation rates due to breast pain/tenderness reported by participants in the SMART trials.17–20 In SMART-5, both the BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg treatment groups demonstrated non-inferiority compared with placebo with regards to mammographic breast density. By contrast, the CE 0.45 mg/MPA 1.5 mg treatment group exhibited a significant increase in breast density compared with the placebo group. BZA/CE treatment did not affect age-related changes in mammographic breast density (ie, natural reduction in breast density throughout the years of menopause). The results demonstrated that, unlike estrogen progestin-containing HT, BZA 20 mg/CE 0.45 mg, and BZA 20 mg/CE 0.625 mg have a neutral (similar to placebo) effect on breast density.20 BODY.SAFETY.GENERAL SAFETY: Based on the integrated safety results of the SMART trials, the BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg dose strengths are well tolerated and have an acceptable safety profile, supporting use in generally healthy, postmenopausal women with a uterus.17–20 The incidences of treatment-emergent adverse events, serious adverse events, and discontinuations due to adverse events were similar among the treatment groups. The incidence of venous thromboembolism was low, as expected in this relatively healthy population and consistent with what was reported in the Women's Health Initiative study.26 The risk does not appear to be any greater than for CE alone or BZA alone or greater than for CE/MPA.26,27 The incidences of coronary heart disease and cerebrovascular accidents were similar to those on placebo. The incidence of estrogen-sensitive cancers, including breast cancer, endometrial cancer, and ovarian cancer, was low and similar to placebo. Clinical laboratory tests demonstrated that the BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg doses were associated with a favorable lipid profile (decreases in total cholesterol, low-density lipoprotein, increases in high-density lipoprotein) with the exception of an increase in triglyceride levels.20 Both the BZA 20 mg/CE 0.45 mg and BZA 20 mg/CE 0.625 mg dose strengths were associated with an increase from baseline in serum triglycerides which was similar to that reported for estrogen progestin-containing HT.28 No other clinically significant changes in clinical laboratory tests were noted. There were no significant differences in the adjusted mean change in body weight with either BZA/CE dose compared with placebo at one year or at the final on-therapy evaluation; this was the case among subjects in the overall population as well as in the two subgroups of women with a BMI <25 kg/m2 or ≥25 kg/m2. The percentages of subjects with body weight changes of potential clinical importance (an increase or decrease from baseline of ≥15% or ≥11 kg) were low and not significantly different between the BZA/CE and placebo groups in the overall population or in the two subgroups of women with a BMI <25 kg/m2 or ≥25 kg/m2.17–20 Changes from baseline in mean systolic blood pressure were less than 2.00 mmHg in the BZA 20 mg/CE 0.45 mg group and BZA 20 mg/CE 0.625 mg group; these changes were similar to those observed for the placebo group. The mean change in systolic blood pressure was comparable with that seen on placebo at all time points evaluated.17–20 BODY.CONCLUSION: Treatment with BZA/CE demonstrated clinically meaningful improvements in vasomotor symptoms and vulvovaginal atrophy as well as a protective effect on the skeleton in postmenopausal women seeking treatment for menopausal symptoms, while protecting the endometrium. BZA/CE also showed significant improvements in tolerability compared with HT, as measured by lower rates of uterine bleeding and breast pain. BZA/CE treatment had an effect on breast density similar to placebo. These clinical benefits were associated with an acceptable safety profile. No apparent increased risk for serious adverse events, such as venous thromboembolism, cardiovascular events, endometrial cancer, or breast cancer, was observed with either dose strength of BZA/CE. BZA/CE is an alternative option for treating non-hysterectomized, symptomatic postmenopausal women.

TITLE: The efficacy of duloxetine: A comprehensive summary of results from MMRM and LOCF_ANCOVA in eight clinical trials ABSTRACT.BACKGROUND: A mixed-effects model repeated measures approach (MMRM) was specified as the primary analysis in the Phase III clinical trials of duloxetine for the treatment of major depressive disorder (MDD). Analysis of covariance using the last observation carried forward approach to impute missing values (LOCF_ANCOVA) was specified as a secondary analysis. Previous research has shown that MMRM and LOCF_ANCOVA yield identical endpoint results when no data are missing, while MMRM is more robust to biases from missing data and thereby provides superior control of Type I and Type II error compared with LOCF_ANCOVA. We compared results from MMRM and LOCF_ANCOVA analyses across eight clinical trials of duloxetine in order to investigate how the choice of primary analysis may influence interpretations of efficacy. ABSTRACT.METHODS: Results were obtained from the eight acute-phase clinical trials that formed the basis of duloxetine's New Drug Application for the treatment of MDD. All 202 mean change analyses from the 20 rating scale total scores and subscales specified a priori in the various protocols were included in the comparisons. ABSTRACT.RESULTS: In 166/202 comparisons (82.2%), MMRM and LOCF_ANCOVA agreed with regard to the statistical significance of the differences between duloxetine and placebo. In 25/202 cases (12.4%), MMRM yielded a significant difference when LOCF_ANCOVA did not, while in 11/202 cases (5.4%), LOCF_ANCOVA produced a significant difference when MMRM did not. In 110/202 comparisons (54.4%) the p-value from MMRM was lower than that from LOCF_ANCOVA, while in 69/202 comparisons (34.2%), the p-value from LOCF_ANCOVA was lower than that from MMRM. In the remaining 23 comparisons (11.4%), the p-values from LOCF_ANCOVA and MMRM were equal when rounded to the 3rd decimal place (usually as a result of both p-values being < .001). For the HAMD17 total score, the primary outcome in all studies, MMRM yielded 9/12 (75%) significant contrasts, compared with 6/12 (50%) for LOCF_ANCOVA. The expected success rate was 80%. ABSTRACT.CONCLUSIONS: Important differences exist between MMRM and LOCF_ANCOVA. Empirical research has clearly demonstrated the theoretical advantages of MMRM over LOCF_ANCOVA. However, interpretations regarding the efficacy of duloxetine in MDD were unaffected by the choice of analytical technique. BODY.BACKGROUND: Treatment effects are often evaluated by comparing change over time in outcome measures. However, valid analyses of longitudinal data can be problematic, particularly if some data are missing for reasons related to the outcome measure [1,2]. Since the problem of missing data is almost ever-present in clinical trials, numerous methods for handling missingness have been proposed, examined, and implemented [3]. A common method of analyzing clinical trial data is to use analysis of variance or analysis of covariance (ANOVA or ANCOVA) with missing data imputed by the last observation carried forward approach (LOCF_ANCOVA). The popularity of LOCF_ANCOVA may be due to its simplicity, and also the belief that violations of the restrictive assumptions inherent to LOCF_ANCOVA lead to a conservative analysis [4]. Considerable advances in statistical methodology, and in our ability to implement these methods, have been made in recent years. Thus, methods that require less restrictive assumptions than LOCF_ANCOVA are now readily implemented. For example, likelihood-based repeated measures approaches have a number of theoretical and practical advantages for analysis of longitudinal data with dropout [4]. One such method, termed MMRM (Mixed Model Repeated Measures [5]), has been studied extensively in the context of neuropsychiatric clinical trials [6-9]. In these studies, MMRM was found to be more robust to biases from missing data than LOCF_ANCOVA, and thereby provided superior control of Type I and Type II errors. The LOCF_ANCOVA method was shown to underestimate treatment group differences in some scenarios, while overestimating differences in others. When no data were missing, the two methods yielded identical results. The MMRM approach was specified as the primary analysis in the Phase III clinical trials of duloxetine for the treatment of major depressive disorder (MDD), while LOCF_ANCOVA was specified as a secondary analysis. In the present investigation, we provide a comprehensive summary of results from MMRM and LOCF_ANCOVA in the eight acute-phase clinical trials that formed the basis for duloxetine's New Drug Application (NDA) for MDD. The primary objective of this investigation was to determine whether differences in results between MMRM and LOCF_ANCOVA influenced conclusions regarding the efficacy of duloxetine. BODY.METHODS.DATA: The data source for this investigation was the eight acute-phase clinical trials in which duloxetine was compared with placebo in the treatment of MDD. Relevant details of these studies are highlighted in Table 1. Table 1 Summary of studies included in the comparisons between MMRM and LOCF_ANCOVA Study Treatment Duration Drug Number of Patients Drug Dose & Design 1 9 weeks Placebo 122 - Duloxetine 123 60 mg/d (QD) 2 9 weeks Placebo 139 - Duloxetine 128 60 mg/d (QD) 3 8 weeks Placebo 70 - Duloxetine 70 40–120 mg/d (20 mg-60 mg BID) Fluoxetine 33 20 mg/d (QD) 4 8 weeks Placebo 75 - Duloxetine 82 40–120 mg/d (20 mg-60 mg BID) Fluoxetine 37 20 mg/d (QD) 5 8 weeks Placebo 90 - Duloxetine 91 40 mg/d (20 mg BID) Duloxetine 84 80 mg/d (40 mg BID) Paroxetine 89 20 mg/d (QD) 6 8 weeks Placebo 89 - Duloxetine 86 40 mg/d (20 mg BID) Duloxetine 91 80 mg/d (40 mg BID) Paroxetine 87 20 mg/d (QD) 7 8 weeks Placebo 93 - Duloxetine 95 80 mg/d (40 mg BID) Duloxetine 93 120 mg/d (60 mg BID) Paroxetine 86 20 mg/d (QD) 8 8 weeks Placebo 99 - Duloxetine 93 80 mg/d (40 mg BID) Duloxetine 103 120 mg/d (60 mg BID) Paroxetine 97 20 mg/d (QD) Results are summarized from all rating scale total scores, subscales, and global assessments that were specified a priori in the various protocols to be analyzed for mean change from baseline to endpoint, and were collected at more than one postbaseline time point (Table 2). Efficacy measures that were assessed only at baseline and endpoint were not included in this summary because repeated measures analyses were not possible for these outcomes. Thus, the present investigation included every rating scale total score and subscale from every clinical trial relevant to duloxetine's NDA for an indication in major depression. In total, 20 efficacy and health outcome variables were included in the summary of MMRM and LOCF_ANCOVA. Some of the eight trials included multiple dose arms; therefore, some outcomes were assessed in as many as 12 comparisons with placebo. Table 2 Outcomes included in the summary of results. 17-item Hamilton Rating Scale for Depression (HAMD 17 )  Total score [24]  Core subscale (items 1, 2, 3, 7, 8) (not published)  Maier subscale (items 1, 2, 7, 8, 9, 10) [25]  Anxiety/Somatization subscale (items 10, 11, 12, 13, 15, 17) [26]  Retardation subscale (items 1, 7, 8, 14) [27]  Sleep subscale (items 4, 5, 6) [27] Montgomery-Asberg Depression Rating scale [28] Hamilton Anxiety Rating Scale total score [29] Clinical Global Impression of Severity [26] Patient Global Impression of Improvement [26] Visual Analog Scale for pain [30]  Overall pain severity  Headaches  Back pain  Shoulder pain  Time in pain while awake  Interference with daily activities Somatic Symptom Inventory [31]  26 Item total score  28 Item total score (includes 2 additional questions on painful physical symptoms) Quality of Life in Depression Scale total score [32] Comparisons of MMRM and LOCF_ANCOVA focused on contrasts between duloxetine and placebo. However, six of the studies also included known effective antidepressants approved for marketing in the United States and other countries. Contrasts between duloxetine and the active comparators are not included in this summary since these results may draw attention to the drug versus drug results and detract from the primary focus of comparing MMRM with LOCF_ANCOVA. BODY.METHODS.STATISTICAL ANALYSIS: This summary makes no attempt to provide formal statistical comparisons of results from MMRM and LOCF_ANCOVA. Previous research has demonstrated conclusively that in the absence of missing data the two methods yield identical endpoint contrasts, while differences do exist in the presence of subject dropout [6-9]. Furthermore, formal statistical comparisons are typically applied to random samples obtained from larger populations in order to assess the uncertainty associated with the sampling. However, the eight studies included in this summary are not a sample, but rather represent all of the acute-phase, double-blind, placebo-controlled trials of duloxetine. Thus, there is no uncertainty associated with sampling. Consequently, results from the two methods need only be summarized in order to assess how differences between the methods may influence overall conclusions regarding the efficacy of duloxetine. Three overall summary measures were used to compare results from the two analytic techniques: 1) With regard to statistical significance of the difference between duloxetine and placebo, the proportion of outcomes showing agreement between MMRM and LOCF_ANCOVA was compared with the proportion of outcomes for which MMRM and LOCF_ANCOVA yielded disparate results; 2) The proportion of outcomes for which MMRM yielded the lowest p-value was compared with the corresponding proportion for LOCF_ANCOVA; 3) The number of outcomes for which "substantial evidence of efficacy" was demonstrated. In regulatory settings, the criterion for substantial evidence of efficacy is frequently the demonstration of a statistically significant advantage over placebo in two or more studies. This criterion was utilized here to define substantial evidence of efficacy for a particular outcome. The frequency of lower p-values provides a "fine-tuned" measure of sensitivity of the two analytic methods. However, in certain cases such an assessment may actually be misleading. For example, to distinguish between p= .800 and p = .810, or between p =. 002 and p =. 003, implies that the methods yielded different results when in fact the similarities far outweigh the differences. Hence, it is equally appropriate to simply categorize based upon the presence or absence of a significant difference. Furthermore, given the large number of outcomes assessed across the eight studies, it would not be surprising to see the two methods disagree with regard to statistical significance on at least a small number of outcomes. Therefore, perhaps the most clinically meaningful summary measure is the number of outcomes for which substantial evidence of efficacy was demonstrated. Three outcomes were selected for more detailed presentation of results: the 17-item Hamilton Rating Scale for Depression (HAMD17) total score, the HAMD17 Maier subscale, and the Visual Analog Scale (VAS) for overall pain severity. The HAMD17 total score was an obvious choice as it was the primary outcome in all studies. The other outcomes were selected since they are frequently focal points in manuscripts and presentations regarding duloxetine's efficacy. Finally, we provide case studies to help explain how and why results from MMRM and LOCF_ANCOVA may differ. MMRM and LOCF_ANCOVA analyses were specified in the Phase III duloxetine protocols as follows. In LOCF_ANCOVA analyses, change from baseline to the last observation was the dependent variable. Treatment and investigative site were included as categorical independent variables, and baseline severity was included as a covariate. In MMRM analyses, change from baseline to all postbaseline times was the dependent variable. Independent variables included the fixed, categorical effects of investigative site, treatment, time, and the treatment-by-time interaction, along with the continuous covariates of baseline severity and the baseline severity by time interaction. Parameters were estimated using Restricted Maximum Likelihood with the Newton-Raphson algorithm. The protocols specified an algorithm for choosing the best fitting covariance structure. In all cases an unstructured matrix provided the best fit. Hence, within-patient errors were modeled using an unstructured covariance matrix. BODY.RESULTS: The protocols for the eight studies in the duloxetine NDA specified a priori a total of 202 mean change analyses for the 20 rating scale total scores or subscales. The frequency of significant outcomes and the frequency of higher/lower p-values for each analytic technique are summarized in Table 3. MMRM and LOCF_ANCOVA agreed with regard to substantial evidence of efficacy for 18 of the 20 outcomes, with each analysis yielding substantial evidence for 15 outcomes. That is, MMRM and LOCF_ANCOVA both found substantial evidence of efficacy for 14 outcomes; both methods did not find substantial evidence for four outcomes; and each method found substantial evidence when the other did not for one outcome (Table 3). Table 3 Summary of results from MMRM and LOCF_ANCOVA in duloxetine clinical trials. Outcome Frequency of significant outcomes 1 MMRM LOCF_ANCOVA P-value lower with MMRM LOCF_ANCOVA Outcome significant with MMRM but not LOCF_ANCOVA Outcome significant with LOCF_ANCOVA but not MMRM HAMD 17  Total score 9\12 6\12 9\12 1\12 3\12 0\12  Core subscale 9\12 8\12 5\12 3\12 2\12 1\12  Maier subscale 10\12 9\12 6\12 2\12 2\12 1\12  Anxiety subscale 6\12 5\12 7\12 3\12 2\12 1\12  Retardation subscale 8\12 6\12 6\12 3\12 4\12 2\12  Sleep subscale 1\12 1\12 10\12 2\12 0\12 0\12 MADRS Total score 5\10 3\10 7\10 2\10 2\10 0\10 HAMA Total score 3\10 3\10 4\10 5\10 0\10 0\10 CGI-Severity 6\12 6\12 8\12 2\12 0\12 0\12 CGI-Improvement 1\2 1\2 2\2 0\2 0\2 0\2 PGI-Improvement 9\12 7\12 8\12 2\12 2\12 0\12 Somatic Symptom Inventory  26-item Average score 2\10 2\10 7\10 3\10 0\10 0\10  28-item Average score 3\10 2\10 7\10 3\10 1\10 0\10 VAS Pain Severity  Overall pain 3\10 4\10 2\10 8\10 1\10 2\10  Headaches 1\10 0\10 5\10 5\10 1\10 0\10  Back pain 2\10 2\10 6\10 4\10 1\10 1\10  Shoulder pain 2\10 1\10 3\10 6\10 2\10 1\10  Daily activities 1\10 0\10 2\10 8\10 1\10 0\10  Pain while awake 2\10 2\10 5\10 5\10 1\10 1\10 QLDS Total score 1\4 2/4 1\4 2\4 0\4 1\4 Totals 84\202 70\202 110\202 69\202 25\202 11\202 1. Some of the eight trials included more than one dose arm. Therefore, an individual outcome could be assessed in as many as 12 comparisons with placebo. In 166/202 outcomes (82.2%), MMRM and LOCF_ANCOVA agreed with regard to the statistical significance of the difference between duloxetine and placebo. In 25 cases (12.4%) MMRM yielded a significant difference whereas LOCF_ANCOVA did not, while in 11 cases (5.4%) LOCF_ANCOVA yielded a significant difference when MMRM did not. Both methods tended to yield significance more frequently in depression rating scales and subscales than in outcomes related to somatic and painful physical symptoms. The studies were generally underpowered for these secondary somatic and pain outcomes owing to the greater variance in changes score for these outcomes. For example, the variance in VAS overall pain severity was approximately nine-fold greater than the variance in HAMD17 total scores, leading to a three-fold greater standard error. In 110 of the 202 outcomes (54.4%) the p-value from MMRM was lower than that from LOCF_ANCOVA, while in 69 cases (34.2%) the p-value from LOCF_ANCOVA was lower than that from MMRM. In the remaining 23 cases (11.4%) the p-values from LOCF_ANCOVA and MMRM were equal when rounded to the 3rd decimal place (usually as a result of both p-values being < .001). More detailed results from the three focus outcomes (HAMD17 total score, HAMD17 Maier subscale, and VAS overall pain) are presented in Table 4. In the case of the HAMD17 total score, the advantage of duloxetine over placebo in mean change from baseline to endpoint from MMRM analyses was greater than the corresponding advantage from LOCF_ANCOVA in 9/12 comparisons (Table 4). In 9/12 comparisons the p-value from MMRM was lower than that from LOCF_ANCOVA, while LOCF_ANCOVA yielded a smaller p-value in one case, and p-values were identical in the two remaining cases. In 3/12 comparisons, MMRM yielded a significant difference when LOCF_ANCOVA did not, but in no instance did LOCF_ANCOVA produce a significant difference when MMRM did not. When averaging results across all eight studies, the advantage of duloxetine over placebo in HAMD17 total score was 2.4 from MMRM analyses compared with 2.0 from LOCF_ANCOVA. Thus, the advantage of duloxetine over placebo, based on LOCF_ANCOVA results, was approximately 83% as large as the advantage from MMRM. Table 4 MMRM and LOCF_ANCOVA analysis of three focus outcomes from duloxetine clinical trials. Study 1 Study 2 Study 3 Study 4 Study 5 Study 6 Study 7 Study 8 Dose 60 mg QD 60 mg QD 60 mg BID 60 mg BID 20 mg BID 40 mg BID 20 mg BID 40 mg BID 40 mg BID 60 mg BID 40 mg BID 60 mg BID Δ p Δ p Δ p Δ p Δ p Δ p Δ p Δ p Δ p Δ p Δ p Δ p HAMD 17 Total Score MMRM 4.9 < .001 2.2 .024 3.1 .009 0.9 .415 1.4 .143 1.5 .116 2.4 .034 3.6 .002 2.2 .001 3.3 < .001 1.4 .045 1.6 .014 LOCF 3.8 < .001 1.7 .048 2.1 .066 0.4 .681 1.2 .222 1.5 .138 2.4 .022 3.1 .003 2.2 .007 3.0 < .001 0.9 .253 1.5 .054 HAMD 17 Maier Subscale MMRM 2.8 < .001 1.6 .003 2.0 .005 0.7 .282 1.2 .037 1.4 .012 1.1 .068 1.7 .005 1.4 < .001 2.0 < .001 0.9 .022 1.2 .002 LOCF 2.3 < .001 1.4 .007 1.0 .030 0.6 .481 1.0 .058 1.3 .012 1.5 .028 1.8 .004 1.4 .001 1.9 < .001 0.7 .090 1.0 .014 VAS Overall Pain Severity MMRM 5.9 .055 4.4 .135 NA NA 0.3 .931 1.2 .731 1.0 .771 7.4 .035 5.5 .085 6.3 .050 5.6 .044 1.3 .625 LOCF 6.9 .019 5.2 .037 NA NA 3.5 .573 3.5 .647 3.2 .710 7.1 .048 6.1 .063 5.6 .086 7.8 .014 5.5 .066 NA = not assessed Similar results were obtained for the Maier subscale. Thus, the advantage of duloxetine over placebo in mean change from baseline to endpoint from MMRM was greater than that from LOCF_ANCOVA in 9/12 comparisons (Table 4). The p-value from MMRM was lower than that from LOCF_ANCOVA in 6/12 comparisons, while LOCF_ANCOVA produced a lower p-value in 2 cases, and p-values were identical in the remaining four cases. In 2/12 comparisons MMRM yielded a significant difference when LOCF_ANCOVA did not, while there was one instance in which LOCF_ANCOVA yielded a significant difference when MMRM did not. Averaged over all eight studies, the advantage of duloxetine over placebo in mean Maier subscale score was 1.5 from MMRM analyses compared with 1.3 from LOCF_ANCOVA. Thus, the advantage of duloxetine over placebo based on LOCF_ANCOVA results was approximately 87% as large as the advantage from MMRM. For VAS overall pain severity, the advantage of duloxetine over placebo from MMRM analyses was greater than the corresponding advantage from LOCF_ANCOVA in 2/10 comparisons (Table 4). The p-value from MMRM was lower than that from LOCF_ANCOVA in 2/10 comparisons, while in the remaining 8 comparisons the p-value from LOCF_ANCOVA was lower than that from MMRM. In 1 comparison MMRM yielded a significant difference when LOCF_ANCOVA did not, while in 2 comparisons LOCF_ANCOVA yielded a significant difference when MMRM did not. Over all eight studies, the average advantage of duloxetine over placebo in VAS overall pain severity was 3.9 from MMRM analyses compared with 5.4 from LOCF_ANCOVA. Thus, the advantage of duloxetine based on LOCF_ANCOVA results was approximately 138% as large as the advantage from MMRM. BODY.RESULTS.CASE STUDIES: Mean changes in HAMD17 total score and VAS overall pain severity from two studies (Studies 1 and 2) are used to further illustrate MMRM and LOCF_ANCOVA (analysis of variance with missing data imputed via last observation carried forward) results. Results from these studies were originally reported by Detke et al [10,11]. In both studies the advantage of duloxetine over placebo in HAMD17 total score tended to increase over time whereas duloxetine's advantage in VAS overall pain was greatest at intermediate visits (Tables 5 and 6). Table 5 MMRM and LOCF_ANCOVA analyses of HAMD 17 total score in Studies 1 and 2. STUDY 1 STUDY 2 MMRM THERAPY Week N Mean change Std.error p-value N Mean change Std.error p-value DULOX 1 121 -2.89 0.36 .435 123 -2.89 0.38 .601 PLACEBO 1 115 -2.50 0.37 136 -2.64 0.36 DULOX 2 112 -5.72 0.49 < .001 109 -5.54 0.48 .071 PLACEBO 2 110 -3.39 0.50 129 -4.43 0.45 DULOX 3 105 -7.37 0.53 < .001 108 -6.82 0.55 .287 PLACEBO 3 103 -4.58 0.54 122 -6.06 0.52 DULOX 5 100 -8.76 0.60 < .001 98 -8.58 0.66 .116 PLACEBO 5 101 -5.74 0.60 111 -7.20 0.62 DULOX 7 91 -9.93 0.64 < .001 89 -10.14 0.69 .008 PLACEBO 7 93 -5.82 0.65 97 -7.69 0.65 DULOX 9 84 -10.91 0.70 < .001 81 -10.46 0.71 .024 PLACEBO 9 89 -6.05 0.69 90 -8.29 0.67 LOCF_ANCOVA DULOX LOCF_ANCOVA 121 -9.47 0.63 < .001 123 -8.75 0.71 .048 PLACEBO LOCF_ANCOVA 115 -5.67 0.66 136 -7.02 0.68 Table 6 MMRM and LOCF_ANCOVA analyses of VAS overall pain severity in Studies 1 and 2. STUDY 1 STUDY 2 MMRM THERAPY Week N Mean change Std.error p-value N Mean change Std.error p-value DULOX 1 120 -2.69 1.97 .181 121 -2.02 1.89 .157 PLACEBO 1 113 1.04 2.04 134 1.38 1.79 DULOX 2 113 -8.20 1.87 .003 108 -6.95 2.04 .003 PLACEBO 2 108 -0.42 1.93 127 0.91 1.90 DULOX 3 105 -10.46 1.95 .005 106 -9.81 1.90 < .001 PLACEBO 3 101 -2.56 2.01 119 -1.59 1.78 DULOX 5 99 -8.68 2.20 .028 95 -9.70 2.05 .011 PLACEBO 5 99 -1.85 2.24 108 -2.92 1.92 DULOX 7 91 -10.25 2.34 .016 87 -8.91 2.12 .254 PLACEBO 7 92 -2.26 2.38 94 -5.76 2.01 DULOX 9 83 -8.68 2.18 .055 80 -10.05 2.24 .135 PLACEBO 9 88 -2.80 2.20 88 -5.65 2.12 LOCF_ANCOVA DULOX LOCF_ANCOVA 121 -8.31 2.09 .019 121 -10.39 2.05 .037 PLACEBO LOCF_ANCOVA 114 -1.40 2.18 134 -5.22 1.94 In the case of the HAMD17 total score, advantages for duloxetine over placebo at endpoint (Week 9) from MMRM in Studies 1 and 2 were 4.86 (p < .001) and 2.17 (p = .024), respectively. The corresponding advantages from LOCF_ANCOVA were 3.80 (p < .001) and 1.73 (p = .048). Although the differences were significant for both methods in both studies, MMRM yielded treatment contrasts that were approximately 25% greater than LOCF_ANCOVA. For VAS overall pain, the advantage of duloxetine over placebo at endpoint from MMRM in Studies 1 and 2 were 5.88 (p = .055) and 4.40 (p = .135), respectively. The corresponding advantages from LOCF_ANCOVA were 6.91 (p = .019) and 5.17 (p = .037). In both studies, the endpoint differences were significant from LOCF_ANCOVA, but not from MMRM. The LOCF_ANCOVA treatment contrasts were approximately 15% greater than those from MMRM. Standard errors from LOCF_ANCOVA were approximately 5% smaller than the Week 9 standard errors from MMRM for both the HAMD17 total score and VAS overall pain. BODY.DISCUSSION: In many areas of clinical research, the impact of missing data can be profound [2,12-14]. Traditional approaches to analyses of data from clinical trials with dropouts, such as LOCF_ANCOVA, have focused on ease of implementation and interpretation. However, simple methods rely upon assumptions that are often unrealistic. For example, LOCF_ANCOVA assumes that patient dropout is completely random, i.e. it is unrelated to the outcome being analyzed. Hence, in an analysis of efficacy data, LOCF_ANCOVA assumes that patients do not drop out due to lack of efficacy. The LOCF_ANCOVA approach also assumes that, for those patients who drop out, their observations would not have changed had they stayed in the trial. When these assumptions do not hold true, estimates of treatment effects and associated standard errors may be biased [2-4,6,7,15-17]. Considerable advances in statistical methodology, and in our ability to implement these methods, have been made in recent years. Methods such as MMRM, which require less restrictive assumptions regarding missing data, may now be easily implemented with standard software [4,5,18,19]. No universally superior approach to analysis of longitudinal data exists. However, a series of studies [6-9] demonstrated empirically what may have been anticipated from statistical theory – namely that the MMRM approach, while providing no guarantee of immunity from bias due to subject dropout, was a sensible analytic choice for many clinical trial scenarios. MMRM has repeatedly been shown to provide adequate control of Type I (false positive) and Type II (false negative) errors in a wide variety of situations modeled after neuropsychiatric clinical trials. In these head-to-head comparisons involving 456,000 data sets, the LOCF_ANCOVA approach did not perform as well as MMRM. We therefore specified MMRM as the primary analysis and LOCF_ANCOVA as a secondary analysis in the Phase III clinical trials of duloxetine in the treatment of MDD. Similar results regarding control of Type I and Type II error for LOCF_ANCOVA and mixed-effects model analyses have been obtained independently [16,20-22]. Furthermore, following an independent investigation of data from two placebo- and active-comparator controlled duloxetine trials, in which treatments were coded A, B, C, etc. to blind analysts to the treatment names, Molenberghs et al [4] concluded that MMRM analysis was a sensible choice for those data. The theoretical differences between MMRM and LOCF_ANCOVA have been summarized [5,18], established empirically [6-9], and proven mathematically [4]. However, we are unaware of any previous investigations of how these differences manifest themselves in efficacy assessments of a new medicinal product. The VAS pain results highlight a limitation of endpoint analyses of any type, namely that they provide only a snapshot view of the response profile. From LOCF_ANCOVA analysis, one can only conclude that drug was superior to placebo at endpoint. However, MMRM analysis reveals that drug had a significant effect early in the trials, but that advantage was somewhat transitory as the placebo group tended to "catch up" over time. In order to understand the response profile of a drug, the entire longitudinal profile should be considered [2]. From MMRM, the entire profile can be assessed from the same analysis that provided the primary result (the contrast at endpoint). In the duloxetine database, results from MMRM and LOCF_ANCOVA were in general agreement regarding substantial evidence of efficacy and frequency of significant differences. However, MMRM tended to be more sensitive to drug-placebo differences for outcomes related to overall depressive symptoms and core emotional symptoms of depression, with mean advantages over placebo that were 10% to 20% greater than LOCF_ANCOVA. However, MMRM did not universally increase duloxetine's advantage over placebo in comparison to results from LOCF_ANCOVA. For example, in somatic and painful physical symptom outcomes, results from LOCF_ANCOVA showed mean advantages over placebo that were approximately 40% greater than that from MMRM. Therefore, while the overall conclusions regarding the efficacy of duloxetine were unaffected by the choice of analytic method, this should not mask the important differences between MMRM and LOCF_ANCOVA. The advantages of MMRM and similar methods over LOCF_ANCOVA have been conclusively demonstrated in many studies and are evident in the duloxetine data. Khan et al [23] compiled a database from FDA summaries of efficacy for all antidepressants approved between 1985 and 2000. Less than half of the studies – which were analyzed using LOCF_ANCOVA as the primary analysis – found significant advantages for drug over placebo. These studies were generally anticipated to have at least 80% power and, if the analysis worked as expected, the success rate would be 80%. Therefore, LOCF_ANCOVA was less sensitive to the drug effects than anticipated. In the duloxetine database, MMRM yielded a 75% success rate for the primary outcome measure (HAMD17 total score), while LOCF_ANCOVA produced a 50% success rate, in comparison to the expected rate of 80%. While many factors may reduce the success rate in Phase III clinical trials, the use of a statistical method with known inflation of Type II error (false negative results) is an obvious suspect. An unduly high rate of false negative results could be especially problematic in early phases of drug development where only one or two chances exist to make the correct decision regarding the efficacy of a drug. It is noteworthy that one of the instances when MMMRM yielded a significant difference on the primary outcome (HAMD17 total score) when LOCF did not was in the Phase II study (Study 3). Also consider that across all therapeutic areas only about 50% of the molecules that enter Phase III testing receive regulatory approval. Many factors may reduce the success rate of Phase III development. However, the use in Phase II of a statistical method with known inflation of Type I error (false positive results) is an obvious suspect. Thus, the unexpectedly low success rate in Phase III is consistent with the conclusion that LOCF_ANCOVA inflates Type I error (as a result of an unduly high rate of false positive results in Phase II). Hence, results from the duloxetine NDA are consistent with research suggesting a move away from LOCF_ANCOVA and other simple analytic techniques to methods such as MMRM that are more robust to the biases from missing data. BODY.CONCLUSION: Important differences exist between MMRM and LOCF_ANCOVA. Research has clearly demonstrated the advantages of MMRM over LOCF_ANCOVA. However, interpretations regarding the efficacy of duloxetine in MDD were unaffected by the choice of analytical technique. BODY.COMPETING INTERESTS: Drs. Mallinckrodt, Raskin, Wohlreich, Watkin, and Detke are employees of Eli Lilly and Company. BODY.AUTHOR'S CONTRIBUTIONS: CHM designed the statistical analyses, and participated in interpretation of data and drafting of the manuscript. JR, MMW, JGW, and MJD participated in interpretation of data and drafting of the manuscript. All authors read and approved the final manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here:

TITLE: Core Decompression and Autologous Bone Marrow Concentrate for Treatment of Femoral Head Osteonecrosis: A Randomized Prospective Study ABSTRACT: The aim of this study was to investigate the safety of injection of bone marrow aspirate concentrate during core decompression and to study its clinical (visual analogue scale; Harris-Hip-score) and radiological outcomes (magnetic resonance imaging). In this prospective and randomized clinical trial we evaluated 24 consecutive patients with non-traumatic femoral head necrosis (FHN) during a period of two years after intervention. In vitro analysis of mesenchymal stem cells was performed by evaluating the fibroblast colony forming units (CFU-Fs). Postoperatively, significant decrease in pain associated with a functional benefit lasting was observed. However, there was no difference in the clinical outcome between the two study groups. Over the period of two years there was no significant difference between the head survival rate between both groups. In contrast to that, we could not perceive any significant change in the volume of FHN in both treatment groups related to the longitudinal course after treating. The number of CFU showed a significant increase after centrifugation. This trial could not detect a benefit from the additional injection of bone marrow concentrate with regard to bone regeneration and clinical outcome in the short term. BODY.INTRODUCTION: Aseptic non-traumatic avascular osteonecrosis of the femoral head is a multi-factorial disease, the etiology of which is not entirely clear. Femoral head necrosis (FHN) most commonly affects young patients, often leading to femoral head collapse and resulting secondary osteoarthritis.1 Around 10% of all THA are performed due to FHN.2 In the early stages of the disease [Association Research Circulation Osseous (ARCO) stage I - II] the treatment aims to preserve the joint and to prevent the collapse of the femoral head. One of the most widely used treatment options is the core decompression by retrograde drilling into the necrotic zone. Systematic reviews verified the significantly better head survival rates compared to non-operative treatment options.2 Based on the data by Mont et al., core decompression leads to clinical success in 53-71% of treated patients leaving room for further therapy improvement. Mesenchymal stem cells and osteoblasts that could potentially induce bone formation have been shown to be decreased in both number and activity in afflicted bone. Therefore the local application of autologous mesenchymal stem cells (MSC) into the necrotic region could stimulate the regeneration of the affected bone. MSCs can be isolated from the mononuclear cell fraction of bone marrow and expanded in-vitro to high-cell numbers using laboratory equipment and then injected into the necrotic zone.3 Bone marrow expansion is subject to restricted regulatory laws in many countries.Introduction of a single-use closed system would allow the surgical team to carry out the concentration procedure in the operating room.4 Hernigou et al. introduced the technique of intra-osseous injection of autologous concentrated bone marrow for the treatment of femoral osteonecrosis and pseudarthrosis.5 They reported on the results of core decompression with additional bone marrow grafting in 145 hips with early stage osteonecrosis (ARCO I - II). At 5 years after surgery, the head survival rate in their study was 93%, which would appear to be a considerable improvement over previous studies. Although this prospective study included an impressive number of patients, a control group and a randomized study protocol were missing. The aim of this study was i) to investigate the safety of additional injection of bone marrow aspirate concentrate (BMAC) during core decompression and ii) to study the clinical (visual analogue scale, VAS; Harris-Hip-Score, HHS) and radiological outcome (magnetic resonance tomography, MRI) in comparison to core decompression only. In addition, in vitro analysis of MSCs was performed by evaluating the fibroblast colony forming units (CFU-F's). BODY.MATERIALS AND METHODS.STUDY DESIGN: In this prospective and randomized clinical trial we evaluated 24 consecutive patients (25 hips) with non-traumatic FHN during a period of two years after intervention. All patients signed the informed consent, the institutional review board of the hospital approved the study; all procedures were performed in accordance with the Declaration of Helsinki. The inclusion criteria for this study were age over 18 years and the presence of stage II femoral head osteonecrosis according to the Association of Research Circulation Osseous (ARCO) classification. Before inclusion into the study all patients were evaluated with two plane radiographs and an MRI of the affected hip. At the day of the procedure patients were randomized in two groups using a simple randomization method based upon sequential patient allocation: core decompression only vs. core decompression with the application of BMAC. For clinical outcome the VAS and HHS were measured. For radiological outcome the volume of the necrotic zone and the stage according to ARCO classification was evaluated with an MRI. The subsequent course of osteonecrosis was evaluated at 12 and 24 months postoperatively. This study was approved by the ethics committee of University of Heidelberg, Germany. BODY.MATERIALS AND METHODS.CORE DECOMPRESSION PROCEDURE: In all patients a core decompression procedure was performed under general anesthesia. Under the guidance of an image intensifier in two planes, three 2.0 mm K-wires (Kirschner wires) were drilled through the major trochanter and along the femoral neck axis into the femoral head, reaching the subchondral necrotic area (2-3 mm from the articular cartilage). The most centrally placed K-wire in the necrotic zone was over drilled using 5 mm trephine as has been previously described in the literature. BODY.MATERIALS AND METHODS.BONE MARROW ASPIRATION, PROCESSING AND INSTILLATION: The harvesting of autologous bone marrow was performed by percutaneous aspiration from the ventral iliac crest (both sides) using a bone marrow biopsy device. The initial volume of harvested marrow was 200 to 220 mL. Intra-operative processing and concentration of stem cells was performed using a Sepax centrifugation device from Biosafe SA (Eysins, Switzerland) with a volume reduction protocol that isolates the nucleated cell (NC) fraction in the buffy coat. After cell segregation, the erythrocytes, nucleated cells and plasma were automatically decanted. 12 mL of bone marrow concentrate suspension was isolated. 2 mL of the final cell concentrate volume (BMAC) was collected for further experimental investigations. 10 mL of BMAC was instilled into the necrotic zone through the canulated trephine drill after removal of the central k-wire. BODY.MATERIALS AND METHODS: The number of MSCs within the native bone marrow and the bone marrow concentrate was analyzed using the CFU-F's. Assuming that each adherent MSC is able to form a fibroblast colony, the number of MSCs within a cell culture could be estimated. Per donor, 2×106 mononucleated cells (MNC) out of the native bone marrow and the BMAC were seeded into three T25 tissue culture flasks each and cultured in standard medium consisting of high glucose DMEM, 20% fetal calf serum (FCS), 1% penicillin/streptomycin, 10 ng/mL recombinant human epidermal growth factor (EGF) and 10 ng/mL recombinant human platelet-derived growth factor (PDGF). After seven days of culturing, cells were washed with phosphate buffered saline (PBS), fixed with 70 % ethanol stained with toluidin blue. Fibroblast colonies were counted manually using a microscope at 25th magnification. (Modified protocol from Castro-Malaspina et al; 1980).6 BODY.STATISTICS: Data were expressed as mean or mean ± SEM. The nominal data of patients' characteristics were tested using the test and χ2 Fisher exact test. The distribution of continuous data was determined using the Kolmogorov-Smirnov test and the appropriate parametric or non-parametric test was selected. Differences within the groups for radiological and clinical outcome data were compared with the Wilcoxon test and between the groups – with Mann-Whitney test. A P<0.05 was considered statistically significant. For radiological evaluation MRI scans of 25 hips before surgery, 20 hips at 1 year and 15 hips at 2 years after operation were available due to the presence of endoprosthesis and other reasons. For longitudinal and transverse comparison of the volume of the osteonecrosis, only data of patients with full radiological observation was analyzed. Calculation of FHN volume was performed using GE Healthcare RIS/PACS software (Milan, Italy). SPSS (SPSS Inc., Chicago, IL, USA) and Graph Pad Prism software (Graph Pad, La Jolla, CA, USA) were used for statistical analysis. BODY.RESULTS: Twenty-five hips of 24 patients with femoral head osteonecrosis were included into this study between 2008 and 2010 and assigned by random to the treatment method (14 in control group, 11 in verum group). All 25 hips were fully evaluated clinically (VAS: preoperatively, 3 months postoperatively, 1 and 2 years after treatment; HHP: preoperatively, 3 months, 1 and 2 years after treatment) and if feasible evaluated radiographically (MRI preoperatively, 1 and 2 years after treatment). Both study populations did not significantly differ in age, gender, side of FHN and risks factors for osteonecrosis (steroid therapy in the patients' history) as shown in Table 1. According to the inclusion criteria all patients suffered from an ARCO II FHN and had no prior trauma. No side effects (hematoma, infection, nerve injury and others) either from the bone marrow aspiration from the pelvic rim or from the injection into the femoral head were observed during the entire study period. BODY.RESULTS.CLINICAL AND FUNCTIONAL OUTCOME: The clinical and functional outcome was measured using standardized scores (VAS; HHS). At the time of inclusion into the study there was no significant difference between the two groups regarding pain and function. Postoperatively, significant decrease in pain associated with a functional benefit lasting the entire observation period was observed. However, there was no difference in the clinical outcome between the two study groups (Figures 1 and 2). BODY.RESULTS.RADIOLOGICAL OUTCOME: Before treatment there was no significant difference between the mean volumes of the osteonecrosis in both groups. In contrast to the clinical outcome, we could not perceive any significant change in the volume of FHN in both treatment groups related to the longitudinal course after treating (Figure 3). Again no statistically significant difference was detected between the groups. FHN progressing to ARCO III or IV was defined as failure of the treatment. This includes all patients with joint replacement (total hip arthroplasty) due to the FHN within two years in both groups. Over the period of two years there was no significant difference between the head survival rate of 8/14 (57%) in the control group, and 7/11 (64%) in the verum group. There was no difference between the two groups with regard to the interval between core decompression with or without BMAC application and THA. BODY.RESULTS.BONE MARROW: The bone marrow was analyzed before and after the centrifugation procedure using the Sepax centrifugation device. Table 2 shows the significant increase in the number of nucleated cells due to the centrifugation step. Additionally, the number of CFU that best represents the number of MSC shows a significant increase (Table 2). BODY.DISCUSSION: Femoral head necrosis is a painful disease usually afflicting young male patients and the natural course of this disease tends to be progressive and ends in secondary osteoarthritis.7 In late stage osteonecrosis (ARCO III and IV), total hip arthroplasty (THA) currently seems to be the best treatment with good functional restoration. However, the young patient age and subsequent expected life span is substantially longer than the currently expected longevity of primary THA. Early recognition of FHN and lastly established less invasive treatment modalities open new possibilities to treat this disease for preservation of the spherical femoral head shape indicating a better outcome and finally,8 avoiding THA. One of these operations is the core decompression procedure9-11 and the literature indicates that it is superior to non-operative therapy.12,13 It has been shown that core decompression leads to significant post-operative pain reduction in early stage FHN.2,14 Nevertheless, in postoperative MRI and patho-morphologic trials it could be shown that no significant repair process in the necrotic area of FHN could be achieved with core decompression alone,15 making it a controversial procedure and calling for additional alternative treatment options. The long term results of this procedure are mainly influenced from the initial stage of the osteonecrosis with best prognosis for ARCO I and worst for ARCO III and IV. The outcome of the non-reversible early stage ARCO II is still under discussion. Therefore we included ARCO II hips in this study only. The injection of mesenchymal progenitor cells into the osteonecrosis seems to improve the outcome of FHN as shown by Hernigou et al.16 The application of ex vivo cultivated stem cells for treatment of diseases of the musculoskeletal system is possible, but currently requires advanced laboratory and technical effort. In addition, this two-step procedure is associated with a higher risk of complications and higher costs. On the other hand, cell therapy systems using centrifugation steps were recently developed which permit intra-operative enrichment of mesenchymal progenitor cells from BMAC simultaneously at the time of core decompression followed by application of the BMAC into the necrotic area of FHN at the end of the same procedure. Simultaneous application of BMAC is praised as an encouraging approach. Hernigou et al. (2002, 2005) inaugurated this treatment option and published a large patient series, that demonstrates an obvious improvement in the head survival rate compared to other literature data with core decompression only.5,16 Lastly, Civinini et al. showed good results of treating early stage femoral head necrosis using injectable calcium and sulphate/calcium bioceramic with bone marrow concentrate.17 The first aim of our investigation was to evaluate the safety of the additional aspiration and injection of autologous bone marrow. In our study we did not encounter any complications resulting from this method of bone marrow transplantation. In particular, there were no infections, excessive new bone formation or tumor induction and there were no local complications at the harvesting side. These findings are in line with published data showing the very low risk of this one step procedure.18 The second aim of our study is the effect of the additional injection of BMAC into the femoral head on the clinical and radiological outcome. We found that significant pain relief and functional improvement could be achieved in both groups at the 2 year follow up independent of the operative procedure (core decompression or core decompression with BMAC application). There are a limited number of studies evaluating pain and functional gain after core decompression, and a direct comparison with these studies is difficult. Rajagopa et al. performed a systematic review of four studies evaluating core decompression of femoral head necrosis and its effect on pain and function of the hip over a minimum of two years postoperatively.19 The results of their analysis showed a variable gain of function after core decompression procedure. One of the articles quoted demonstrated a significant improvement in hip function,20 a second one just moderate improvement21 and two other studies only minor, or no improvement.22,23 Our study supports the latter. We did not note any significant differences relating to pain or functional state of patients between both groups, supporting the hypothesis, that additional BMAC application after core decompression provides no benefit to patients with FHN at this time. This finding seems to be in contrast to the studies of Hernigou et al. However, the study presented here was set up as a randomized controlled trial with restrictive inclusion criteria (ARCO II only). The rejection of ARCO I patients with a better prognosis might, at least in part, explain differences to the results of Hernigou et al. and other studies. Table 3 shows a comparison of head survival rate of this study and recently published studies using BMAC or similar bone marrow treatment options in femoral head necrosis. Because of different study protocols, meaningful comparison of these research findings is limited. A study with a protocol similar to ours was published by Gangji et al.24 They presented the results of a randomized and prospective clinical trial of ARCO I-II patients treated with core decompression only vs. core decompression with autologous bone marrow application. Femoral head survival rate of this study is also mentioned in Table 3. Gangji et al. also describe an increased head survival rate in the BMAC group. Although this is a randomized controlled prospective trial, there are some principal limitations to this study: there is a short follow-up period (two years) and there are a small number of patients in both study groups. The small study population is a result of the prospective design and very restrictive inclusion criteria of FHN patients (ARCO II only). In comparison to the study presented by Hernigou et al.,16 the major advantage of this study is the comparison of a BMAC group with a control group, thereby increasing explanatory power. BODY.CONCLUSIONS: Femoral head necrosis with a spherical head and irreversible necrosis of the bone (ARCO II) profits from core decompression. In contrast to previous series the current study excluded ARCO I stages of FHN which can regenerate on its own, and inclusion of ARCO I makes interpretation of drilling of this stage difficult. This trial of 25 hips could not detect a benefit from the additional injection of bone marrow concentrate with regard to bone regeneration and clinical outcome in the short term. Further studies of BMAC properties with a larger sample size and longer follow-up are needed to better validate our results and possibly modify our procedure.

TITLE: The effect of hip abduction on the EMG activity of vastus medialis obliquus, vastus lateralis longus and vastus lateralis obliquus in healthy subjects ABSTRACT.STUDY DESIGN: Controlled laboratory study. ABSTRACT.OBJECTIVES: The purposes of this paper were to investigate (d) whether vastus medialis obliquus (VMO), vastus lateralis longus (VLL) and vastus lateralis obliquus (VLO) EMG activity can be influenced by hip abduction performed by healthy subjects. ABSTRACT.BACKGROUND: Some clinicians contraindicate hip abduction for patellofemoral patients (with) based on the premise that hip abduction could facilitate the VLL muscle activation leading to a VLL and VMO imbalance ABSTRACT.METHODS AND MEASURES: Twenty-one clinically healthy subjects were involved in the study, 10 women and 11 men (aged X = 23.3 ± 2.9). The EMG signals were collected using a computerized EMG VIKING II, with 8 channels and three pairs of surface electrodes. EMG activity was obtained from MVIC knee extension at 90° of flexion in a seated position and MVIC hip abduction at 0° and 30° with patients in side-lying position with the knee in full extension. The data were normalized in the MVIC knee extension at 50° of flexion in a seated position, and were submitted to ANOVA test with subsequent application of the Bonferroni multiple comparisons analysis test. The level of significance was defined as p ≤ 0.05. ABSTRACT.RESULTS: The VLO muscle demonstrated a similar pattern to the VMO muscle showing higher EMG activity in MVIC knee extension at 90° of flexion compared with MVIC hip abduction at 0° and 30° of abduction for male (p < 0.0007) and MVIC hip abduction at 0° of abduction for female subjects (p < 0.02196). There were no statistically significant differences in the VLL EMG activity among the three sets of exercises tested. ABSTRACT.CONCLUSION: The results showed that no selective EMG activation was observed when comparison was made between the VMO, VLL and VLO muscles while performing MVIC hip abduction at 0° and 30° of abduction and MVIC knee extension at 90° of flexion in both male and female subjects. Our findings demonstrate that hip abduction do not facilitated VLL and VLO activity in relation to the VMO, however, this study included only healthy subjects performing maximum voluntary isometric contraction contractions, therefore much remains to be discovered by future research BODY.INTRODUCTION: Patellofemoral pain syndrome (PFPS) presents one of the most perplexing pathologic conditions in orthopedic and sports medicine clinics, as well as in rehabilitation departments, and it was referred to by Dye [1] as the "black hole of orthopedics" because of the lack of clarity regarding the etiological factors that contribute to dysfunction or to specific treatment protocols and the causative mechanisms remain imprecisely defined [2]. Dysfunction of the quadriceps muscle has been hypothesized as a cause of patellofemoral pain syndrome (PFPS) with great emphasis placed on the role of VMO and VL muscular imbalance [3,4]. Quadriceps dysfunction in PFPS patients has been assessed in various ways including decreased magnitude of the electromyographic (EMG) activity of the quadriceps [5,6], diminished EMG activity of the VMO in relation to that of the VL [7-9], and the delayed onset of VMO activation in relation to the VL [10-12] caused by the inhibition of pain, effusion and atrophies[13]. Improved control of patella tracking is necessary for symptomatic relief [14] and the recovery of quadriceps function is essential to the resolution of the problem [15]. Consequently, there have been numerous studies that have sought to identify exercises to selectively recruit the VMO in an effort to retrain this muscle [7,16-18]. However, Mirzabeigi et al. [19] suggested that the VMO muscle cannot be significantly isolated during nine sets of different exercises. Since fibers of the VMO attach to the adductor magnus muscle, it has been hypothesized that activation of the VMO may be enhanced by combining active knee extension with volitional hip adduction [4,16,20]. Research in clinical studies on the treatment of PFPS has reported that VL activity may also be enhanced by combining knee extension exercises with hip abduction [21,22]. Fulkerson [23] noted retinacular tenderness in patients with patellofemoral pain, including some who demonstrated histological changes within the perineural tissues of the lateral retinaculum on examination of surgical biopsy specimens. Bevilaqua-Grossi et al. [24] in their anatomical study dissected the thighs of 32 human cadavers and showed that the distal fibers of the Vastus Lateralis Obliquus (VLO) were interdigitated with the lateral retinaculum and the iliotibial tract in all specimens, were subsequently joined in a common tendon with the VLL on the superolateral border of the patella (Figure 1) and that tightness of lateral retinaculum could potentially alter the tracking of the patella in the trochlear groove. Based on this anatomic correlation, Hip abduction exercises are often contraindicated by physical therapists because some group of patient with patellofemoral problems may have tight lateral structures and hip abduction exercises would enhance VMO and VL muscular imbalance [21]. Figure 1Lateral view of the right thigh showing the origin of the obliquus portion of the vastus lateralis muscle (vastus lateralis obliquus – VLO) in the lateral intermuscular septum (LIS) and its insertion in the superior -lateral border of the patella (P). VLL – vastus lateralis longus. Bevilaqua-Grossi et al. (2004) 46. Considering that previous few studies have investigated the EMG relationship VMO and VLO with other structures of the lateral compartment (lateral retinaculum and iliotibial tract) and hip abduction strengthening exercises, the aim of this paper was to analyze the EMG activity of the VMO, VLL and VLO muscles and verify whether any difference in activity between these portions occurred during MVIC: 1) knee extension at 90° of flexion, 2) hip abduction at 0° of abduction and 3) hip abduction at 30° of abduction. The data reported in this paper should be useful in future functional studies aimed at a clearer understanding of rehabilitation protocols in PFPS patients. BODY.METHODS.SUBJECTS: Twenty-one healthy volunteers (11 males and 10 females), aged from 19 to 28 (X = 23.3 ± 2.9), participated in this study. They were recruited from Piracicaba Methodist University and all reported no history of orthopedic disorders, surgical procedures, knee pain or other major musculoskeletal injuries. Prior to participation, all subjects read, accepted and signed a consent form that was approved by the Human Research Ethics Committee at the State University of Campinas. BODY.METHODS.INSTRUMENTATION: Silver/silver chloride surface electrodes placed in a bipolar configuration, with a 10 mm contact area and an inter-electrode distance of 2 cm, were used to assess the level of electromyographic activity of the VMO, VLL and VLO muscles. Before the electrode placement the sites were prepared by shaving, abrading and cleaning with isopropyl alcohol to reduce the surface impedance to less than 5 and 10 kΩ for men and women, respectively. A quadriceps line was drawn from the anterior superior iliac spine to the center of the patella for the quadriceps portion placement [24]. A surface electrode for the VMO was placed with a medial inclination of 55° from the quadriceps line [25,26]. The VLL electrode was placed 15 cm from the superior edge of patella at a lateral inclination of 13.6° and the VLO shows its superficial part around 2.2 cm of the lateralis epicondyle with a superficial length of around 8.9 cm with a 50.4° of lateral inclination[27]. A ground electrode was placed over the tibial tubercle of the tested lower limb. The same investigator performed all electrode placements. A calibrated Viking II with eight channels (Nicolet Biomedical Instruments) and a computer were used to collect all EMG data (CMRR of 110 dB, sampling 1000 Hz, gain 200, band pass filter of 10 to 1000 Hz). All signals were viewed on a display screen prior to collection to ensure that there were no visible artifacts. BODY.METHODS.PROCEDURES: Following EMG preparation, the subjects were instructed to perform 3 repetitions of the following three sets of exercises: 1) MVIC knee extension at 90° of flexion in a seated position; 2) MVIC hip abduction at 0° of abduction, with patients in side-lying position with the knee in full extension; 3) MVIC hip abduction at 30° of abduction, with patients in side-lying position with the knee in full extension During the MVIC knee extension test, the subjects were positioned seated in a leg extension machine (Queens, São Paulo, BRA) with the knee and hip at 90° of flexion and the ankle in a neutral position (figure 2). For the MVIC hip abduction (0° and 30°) tests, the subjects were positioned on their sides lying on a divan with the test lower limb placed above and with both lower limbs positioned at neutral hip and knee flexions, as measured by the investigator. For the maintenance of these positions the thighs were stabilized with padding and the calves were fixed with a belt applied immediately distal to the knees (Figure 3 and 4). Figure 2Maximum voluntary isometric contraction at 90 degrees of knee extension. Figure 3Maximum voluntary isometric contraction of hip abduction in neutral in side-lying position. The subjects were instructed to maintain an isometric quadriceps contraction at full knee extension while performing the task. Figure 4Maximum voluntary isometric contraction at 30 degrees of hip abduction in side-lying position. The subjects were instructed to maintain an isometric quadriceps contraction at full knee extension while performing the task. Before data collection procedures began, each subject received a verbal explanation and a demonstration of the testing activities and practice trials were performed to ensure the subject's comprehension and safety. After familiarization, the subjects randomly performed three MVIC for 5 seconds with a 2 minute rest between repetitions and 10 minutes between each set of exercises to prevent muscle exhaustion. EMG signals were collected throughout each MVIC and verbal encouragement was provided throughout the testing. BODY.METHODS.DATA ANALYSIS: The normalization of the VMO, VLL and VLO EMG signals for the three exercises (knee extension at 90° of flexion, MVIC hip abduction at 0° of abduction, MVIC hip abduction at 30° of abduction) were obtained dividing the highest EMG value of the MVIC trials by the EMG value of a MVIC knee extension at 50° of flexion in a seated position and multiplied by 100 [16]. The normalization procedure performed at 50° of knee flexion in a seated position followed the same protocol of positioning for the test at 90° of knee flexion in a seated position. Normalized EMG readings were analyzed by two-way analysis of variance with repeated measurements. Post hoc comparisons within the values obtained for each of the muscles were realized by the Bonferroni multiple comparisons analysis. Significance was defined as p ≤ 0.05. BODY.RESULTS: Maximum voluntary isometric contractions for knee extension at 90° of flexion resulted in a significantly higher EMG activity for the VMO muscle compared with hip abduction at 0° and 30° of abduction for both male (p < 0.008) and female (p < 0.0005) subjects (Table 1, Figure 5). Table 1 Summarized results of normalized EMG values of VMO, VLL and VLO muscles during MVIC knee extension at 90° of flexion and MVIC hip abduction at 0° and 30° of abduction (N = 21) The results are shown as a percentage of MVIC at 50° of knee flexion in a seated position Test procedure VMO (%) VLL (%) VLO (%) Hip abduction at 30° Male 53.70 88.31 75.70 Female 96.07 119.61 103.98 Hip abduction at 0° Male 69.05 110.62 90.25 female 87.09 100.13 92.30 MIVC at 90° of knee flexion Male 144.91 c 115.31 146.72 d female 165.97 b 141.30 162.61 a  p ≤ 0,05 Letters demonstrate higher EMG activity of the same muscle in which there were statistically significantly differences between the three exercises a p = 0,02196 High EMG activity MVIC knee extension at 90° of flexion vs MVIC hip abduction at 0° of abduction b p = 0,008 High EMG activity MVIC knee extension at 90° of flexion vs MVIC hip abduction at 0° and 30° of abduction c p = 0,000055 High EMG activity MVIC knee extension at 90° of flexion vs MVIC hip abduction at 0° and 30° of abduction d p = 0,000761 High EMG activity MVIC knee extension at 90° of flexion vs MVIC hip abduction at 0° and 30° of abduction Figure 5Normalized EMG activity expressed as RMS values from healthy subjects performing MVIC knee extension at 90° of flexion, MVIC hip abduction at 0° and 30° of abduction. A) Significantly higher VMO activity for both male and female subjects performing MVIC knee extension at 90° of flexion when compared to MVIC hip abduction at 0° and 30° of abduction. B) VLL EMG activity without significant differences among the three exercises tested. C) Significantly higher VLO activity for male subjects was observed in MVIC knee extension at 90° of flexion compared with MVIC hip abduction at 0° and 30° of abduction and for female subjects performing MVIC knee extension at 90° of flexion when compared with MVIC hip abduction at 0° and 30° of abduction. There were no statistically significant differences in the VLL EMG activity among the three types of exercise tested (Table 1, Figure 5). The VLO muscle demonstrated a similar pattern to the VMO muscle showing higher EMG activity in MVIC knee extension at 90° of flexion compared with MVIC hip abduction at 0° and 30° of abduction for male (p < 0.0007) and with MVIC hip abduction at 0° of abduction for female subjects (p < 0.02196) (Table 1, Figure 5). There were no significant differences between gender with respect to the EMG activity of the VMO, VLL and VLO muscles among the three types of exercise tested. No selective EMG activation was observed when comparison was made between the VMO, VLL and VLO muscles while performing MVIC knee extension at 90° of flexion and MVIC hip abduction at 0° and 30° of abduction for both male and female subjects. BODY.DISCUSSION: The primary purpose of this article was to investigate whether VMO, VLL and VLO EMG activity can be influenced by hip abduction. The results showed that no selective EMG activation was observed when comparison was made between the VMO, VLL and VLO muscles while performing MVIC knee extension at 90° of flexion or MVIC hip abduction at 0° and 30° of abduction for both male and female subjects. These results are in agreement with Hertel et al. [4] who investigated the EMG activity of the VMO, VLL and gluteus medius in eight healthy young adult volunteers with no history of knee injury while performing uniplanar knee extension, knee extension/hip adduction, knee extension/hip abduction and found no significant differences in the VMO/VL ratio between the exercises. It is well established in the literature that the isolation of any of the quadriceps components or selective strengthening is unlikely, especially concerning the VMO/VLL muscles [28,29]. Our findings demonstrate that hip abduction do not facilitated VLL and VLO activity in relation to the VMO in healthy subjects performing maximum voluntary isometric contraction. We are unaware of any studies which have demonstrated the characteristics of EMG activity between VMO, VLL and VLO in subjects performing hip abduction in different positions. The secondary purpose of the present work was investigating the EMG activity of the VMO, VLL and VLO while performing the three exercises tested. The results showed that the VLO presents a significantly different behavior compared with the VLL, whereas the VLO showed a similar motor unit recruitment to the VMO, producing higher EMG activity in MVIC knee extension at 90° of flexion compared with MVIC hip abduction at 0° and 30° of abduction. No significant differences were found in the EMG activity of the VLL among the three exercises tested, thus verifying that the VLL and VLO demonstrate not only anatomical but distinct motor unit recruitment characteristics. Bevilaqua-Grossi et al. [27] investigated the EMG activity of the VMO, VLL and VLO muscles in 21 healthy subjects performing open kinetic chain knee extension at 15° and 90° of flexion. The results showed that the VLO and VMO were more active at 90° of flexion compared with the higher activity of the VLL at 15° of flexion, demonstrating that the VMO and VLO muscle have the same behavior suggesting a synchronic antagonist stability role of the patella in healthy people. The striking difference between VLL and VLO behavior concerning the EMG activity observed, could be because the VLL fiber alignment tends to traction the patella, offering greater contribution to knee extension than patella stabilization, different from the VLO which spirally and inclination fibers in relation to femoral dyaphisis promotes patella alignment associated with the VMO [24] Hertel et al. [4] reported that both the VMO and VL are more activated in uniplanar knee extension when compared with knee extension/hip adduction or abduction. These results are not entirely supported by our results, in which the VLL showed no significant differences between MVIC knee extension at 90° of flexion compared with MVIC hip abduction at 0° and 30°. These conflicting results concerning the VLL recruitment pattern may be because these authors tested the exercises in closed kinetic chain while the three exercises tested in the current work were performed in open kinetic chain. Possible limitations of this study may be related to the non collection of gluteus medius EMG activity during the three exercises tested. Normalization methods using other types of muscle contractions or other angles of the knee joint may result in different VMO/VLL/VLO ratios between studies that use different methods of normalization. The absence of increased EMG activity with hip abduction could have been due limitations in EMG recordings from MVCs. Results might be different when studying submax voluntary contractions. Although normalized EMG data are useful in measuring relative levels of activity between muscles, such information is not indicative of muscular strength or muscular balance [58]. Future studies of this research group will focus on subjects with patellofemoral pain and investigate the recruitment patterns provided from such subjects performing the same task. BODY.DISCUSSION.CLINICAL IMPLICATIONS: Bevilaqua-Grossi et al. [24] dissected the thighs of 32 human cadavers and determined the anatomical organizations of the VLL and VLO muscles. The distal fibers of the VLO were interdigitated with the lateral retinaculum and the iliotibial tract in all specimens, which subsequently joined in a common tendon with the VLL on the superolateral border of the patella. These results agree with previous anatomical studies which describe the origin [30-34] and insertion of these muscles [35,36]. Thus, tightness of the lateral retinaculum, perhaps as a result of increased tension in the iliotibial tract, could potentially alter the tracking of the patella in the trochlear groove, becoming an important factor in the etiology of patellofemoral pain [37]. Following this theory, some clinicians contraindicate rehabilitation exercises using hip abduction in patients with patellofemoral complaints based on the premise of avoiding excessive tightness of the lateral structures or a VLL and VMO imbalance, since the anatomical origin of the iliotibial tract has a close relation with the iliotibial band and the gluteus medius muscles [21,38]. This theory is not supported by recent works which investigated the role of pelvic control as a contributing factor in the development of anterior knee pain [39-42]. Ireland et al. [39], using hand held dynamometers, investigated hip abduction and external rotation isometric strength in 15 female patients with patellofemoral pain compared with a control group. They found significant weakness of the hip abductors and external rotators of the patellofemoral pain group. It is postulated that in the absence of pelvic control due to hip abductor and external rotator weakness, the femur may adduct and internally rotate, further increasing lateral patellar contact pressure [43]. These findings suggest that hip abduction exercises may be indicated and necessary for patients with patellofemoral pain who present absence of satisfactory pelvic control. BODY.CONCLUSION: The results showed that no selective EMG activation was observed when comparison was made between the VMO, VLL and VLO muscles while performing MVIC at 30° and 0° of hip abduction and 90° of knee flexion for both male and female subjects. Our findings demonstrate that hip abduction do not facilitated VLL and VLO activity in relation to the VMO, however, this study included only healthy subjects performing maximum voluntary isometric contraction contractions, therefore much remains to be discovered by future research

TITLE: Antioxidative and antiatherogenic effects of flaxseed, α-tocopherol and their combination in diabetic hamsters fed with a high-fat diet ABSTRACT: Oxidative stress has previously been shown to play a role in the pathogenesis of diabetes mellitus (DM) and its complications. In the present study, the effects of supplementation with dietary antioxidants, flaxseed and α-tocopherol were investigated in diabetic golden Syrian hamsters fed with a high-fat diet. Thirty-five golden Syrian hamsters were randomly divided into a control group (C) and four diabetic groups (DM, DM + flax, DM + E and DM + Flax + E). The hamsters received four different diets for a 20-week period, as follows: i) Groups C and DM received a high-fat diet (40% energy as fat), deficient in α-linolenic acid (ALA); ii) the DM + Flax group received a high-fat diet enriched with ground flaxseed 15 g/100 g of food, rich in ALA; iii) the DM + E group received a high-fat diet enriched with vitamin E, 40 mg α-tocopherol/100 g of food; and iv) the DM + Flax + E group received a high-fat diet enriched with flaxseed and vitamin E. The results of serum lipid and oxidative stress analysis suggested that the antiatherogenic effect of flaxseed, α-tocopherol and their combination added to a high-fat diet in diabetic hamsters was based primarily on their antioxidative role, demonstrated by decreased serum lipid peroxidation and increased liver glutathione content. Improvements of serum glucose and non-high-density lipoprotein cholesterol (HDL-C) levels were observed and may have contributed to the prevention of diabetic macroangiopathy evidenced in the histopathological examination. The antioxidant effect of flaxseed was similar to that of α-tocopherol in diabetic hamsters fed a high-fat diet and combined supplementation did not appear to bring more benefits than flaxseed alone. Moreover, the high dose of ground flaxseed alone may have a better cardioprotective effect than α-tocopherol in diabetic hamsters by reducing total cholesterol and non-HDL-C levels and increasing HDL-C levels. BODY.INTRODUCTION: Oxidative stress is considered to be significant in the pathogenesis of diabetes-induced cardiovascular disease (CVD), which is associated with an abnormal blood lipid profile, insulin resistance and metabolic syndrome (1). Oxidative stress occurs due to a disturbance of the balance between antioxidant defense mechanisms and the levels of reactive oxygen species (ROS) (2,3). Numerous studies have identified significant alterations in plasma antioxidant vitamins (including vitamins C, E and A), antioxidant enzyme systems [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px)] and in lipid peroxidation in diabetes (4,5). In chronic diabetes mellitus (DM), the stability and reactive capacity of antioxidants seriously affect the long-term complications caused by oxidative stress (6,7). Antioxidants have been shown to reduce complications in DM by preventing free radical-mediated damage (4,8). Dietary supplementation with antioxidants, including vitamins and phenolic compounds obtained from plants, may help to maintain a desirable pro-oxidative/antioxidative balance (9,10). A variety of dietary sources are presently of considerable interest due to their potential health benefits in relation to numerous diseases, including diabetic disorders (11,12). Flaxseed (also known as linseed and Linum usitatissimum), an edible oil seed/grain from a traditional arable crop, has been acknowledged as a functional food (13), and is the focus of considerable attention due to its unique nutrient components and its potential to prevent CVD (14). In addition of being one of the richest plant sources of α-linolenic acid (ALA; ~22% of whole flaxseed), flaxseed is an essential source of lignans (in particular, secoisolariciresinol diglucoside, SDG) (15). SDG has demonstrated extreme radical scavenging activity, thereby protecting polyunsaturated fatty acids from oxidation (16–18). Despite the beneficial effects of SDG, flaxseed consumption has been found to significantly reduce α- and γ-tocopherol concentrations in rats (19) and only vitamin E supplementation was able to restore the plasma concentrations (20). α-tocopherol supplementation has previously been demonstrated to be beneficial in increasing the levels of antioxidant enzymes such as SOD and GSH-Px, and of reduced GSH in diabetes (21–23); however, little is known about the effect of adding vitamin E to a flaxseed-enriched diet on oxidative stress and aortic atherosclerotic lesions in diabetes. In the present study, the effects of dietary flaxseed, α-tocopherol and their combination were investigated on atherosclerotic progression in diabetic hamsters fed a high-fat diet by examining serum lipid concentrations, oxidative stress markers, aortic cholesterol content and aortic histological aspects. BODY.MATERIALS AND METHODS.ANIMALS: A total of 35 male Golden Syrian hamsters were used in the study. The hamsters were purchased from the Cantacuzino National Institute for Research and Development in Microbiology and Immunology (Bucharest, Romania). This study was approved by the Laboratory Animal Care Committee of Grigore T. Popa University of Medicine and Pharmacy (Iaşi, Romania), and the hamsters were maintained in accordance with the general guidelines for the care and use of laboratory animals recommended by the Council of European Communities (24). The hamsters were caged singly and kept in standard laboratory conditions with a controlled temperature (20±2°C) and a 12 h light/12 h dark cycle. Diabetes was induced in the hamsters using streptozotocin (STZ; catalog no. S0130; Sigma-Aldrich, St. Louis, MO, USA) (25). STZ, at a dose of 50 mg/kg body weight, freshly solved in citrate buffer (50 mM, pH 4.5), was administered intraperitoneally, as a single dose in the morning, following a night of fasting. Citrate buffer (50 mM) was prepared from sodium citrate and citric acid (S.C. Chemical Company, Iaşi, Romania) dissolved in distilled water, with adjustment of the pH to 4.5. The control group (C) received an equivalent quantity of citrate buffer (50 mM), administered intraperitoneally. All animals having a blood glucose level >126 mg/dl (7 mmol/l) were considered to be diabetic. BODY.MATERIALS AND METHODS.GROUPING AND DIETS: Each hamster received 15 g food/day and tap water ad libitum. The animals were randomly divided into a control group (C) and four diabetic groups (DM, DM + flax, DM + E and DM + Flax + E). The hamsters received four different diets for a 20-week period, as follows: i) Groups C and DM received a high-fat diet (40% energy as fat), that was deficient in ALA; ii) the DM + Flax group received a high-fat diet enriched with ground flaxseed (Linum usitatissimum; 15/100 g of food), that was rich in ALA; Flaxseed of the Olin variety was provided by the Department of Phytotechny of the Faculty of Agronomy (Iaşi, Romania). iii) the DM + E group received a high-fat diet enriched with vitamin E (40 mg α-tocopherol/100 g of food; Sigma-Aldrich); and iv) the DM + Flax + E group received a high-fat diet enriched with a combination of flaxseed and vitamin E. All four diets had similar carbohydrate, total fiber, protein and fat contents (Table I). BODY.MATERIALS AND METHODS.ANIMAL NECROPSY AND PROCESSING OF SAMPLES: At the end of the experiment, the animals were sacrificed by cardiac puncture under ketamine anesthesia (100 mg/kg body weight). Blood samples were collected without anticoagulant. Aliquots of serum were frozen and kept at −80°C for subsequent analysis. The liver was immediately removed, rinsed with ice-cold saline, placed in a sealed container, homogenized for biochemical analysis and stored at −20°C until analyzed. The aortas were dissected, rinsed with cold saline and preserved in a phosphate buffer (pH 7.2). BODY.MATERIALS AND METHODS.SERUM GLUCOSE: Serum glucose was measured by enzymatic colorimetric methods on a Tecan microplate reader (Tecan SunriseTM Touchscreen, Männedorf, Switzerland) using the AD1A716 commercially available kits (Audit Diagnostics, Cork, Ireland). BODY.MATERIALS AND METHODS.SERUM LIPID PROFILE: Serum total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) levels were measured by enzymatic colorimetric methods on a Tecan microplate reader using the AD1A704, AD1A602 and AD1A306 commercially available kits (Audit Diagnostics). Non-HDL-C levels were calculated by subtracting the level of HDL-C from that of TC. BODY.MATERIALS AND METHODS.EVALUATION OF CHOLESTEROL CONTENT IN THE AORTA: The aortas of the hamsters in each group were removed. A section of the aortic arch of each animal was soaked in a 10% (v/v) formal saline solution and was stored on ice prior to being stained and fixed. The remainder of the aorta was soaked in phosphate-buffered saline (PBS) and homogenized for biochemical analysis. The lipid content was extracted by treatment with chloroform and methanol followed by centrifugation (26). The extracted lipid was dissolved in ethanol and measured using a AD1A704 cholesterol assay kit (Audit Diagnostics) utilizing absorbance spectrophotometry (Microlab 300, Vital Scientific, Spankeren, Netherlands) at 500 nm wavelength, and the amount quantified as μg/mg wet tissue. BODY.MATERIALS AND METHODS.PARAMETERS OF OXIDATIVE STRESS: Serum and liver thiobarbituric acid reactive substances (TBARS) are indices of lipid peroxidation and oxidative stress. TBARS were determined using the method described by Phelps and Harris (27). The quantity of the TBARS was measured using a Tecan microplate reader at a wavelength of 540 nm. Liver levels of reduced GSH were determined by an enzymatic reaction, based on the oxidation of GSH by 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), in the presence of GSH reductase and methylenetetrahydrofolate reductase, monitored at a wavelength of 405 nm (28). Liver SOD levels were determined using the method described by Minami and Yoshikawa (29). BODY.MATERIALS AND METHODS.MORPHOLOGICAL STUDY OF THE AORTA: For evaluation of aortic atherosclerotic lesions by light microscopy, frozen sections of 10 μm thickness were taken from the region of the proximal aorta. The extent of atherosclerosis was visually assessed after cutting the frozen sections at a size of 8–10 mm, fixing them in formalin, briefly washing them with running tap water for 1–10 mins and rinsing with 60% isopropanol prior to staining the sections with Oil Red O (3 mg/ml Oil Red O in 60% acetone and 40% water; Sigma-Aldrich). The samples were then counterstained with hematoxylin and the samples were examined under a light microscope (Olympus BX40, Olympus, Tokyo, Japan). BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Data are expressed as the mean ± standard deviation (SD). Univariate statistical analysis was performed using the Student's t-test and Bonferroni's multiple comparison test (Statistical Software Package SPSS®, version 13, SPSS Inc., Chicago, IL, USA). BODY.RESULTS.SERUM GLUCOSE: The addition of flaxseed or/and vitamin E to the high-fat diet led to significant reductions of serum glucose levels compared with those in the DM group (Table II). BODY.RESULTS.SERUM LIPID PROFILE PARAMETERS: Diabetes resulted in significant increases in the serum concentrations of TC, TG and non-HDL-C, and a significant reduction of HDL-C concentrations compared with those in the control group. Flaxseed supplementation significantly decreased serum TC and non-HDL-C levels and increased HDL-C levels (Table II) compared with those in the unsupplemented DM group. The addition of vitamin E to the flaxseed-rich diet significantly decreased TC, non-HDL-C and TG levels but did not improved the HDL-C level in diabetic hamsters. BODY.RESULTS.CHOLESTEROL CONTENT OF THE AORTA: Aortic cholesterol contents were significantly increased in the DM group fed with a standard diet compared with those in the control group (Table II). Flaxseed or/and vitamin E supplementation of the diet significantly decreased the aortic cholesterol content in the hamsters with DM (Table II). BODY.RESULTS.PARAMETERS OF OXIDATIVE STRESS: The main findings are summarized in Table III. Serum and liver TBARS, as indices of lipid peroxidation, were significantly increased in the DM group compared with those in the control group, while the addition of flaxseed or a combination of flaxseed and α-tocopherol to the high-fat diet of the hamsters with DM significantly decreased these two markers compared with those in the unsupplemented DM group. Liver GSH levels were significantly diminished in the DM group compared with those in the control group. Flaxseed, α-tocopherol and their combination added to the high-fat diet significantly increased the liver GSH levels in the diabetic hamsters. The increased in liver GSH content was significantly higher in the DM + Flax + E group compared with that in the DM + Flax group. No significant differences of SOD activity in the liver were identified between groups. BODY.RESULTS.HISTOLOGICAL CHANGES IN AORTIC TISSUES.CONTROL GROUP: The histologic examination of aortal fragments with O-Rd staining revealed discrete thickening of the aortic intimal layer (Fig. 1A). BODY.RESULTS.HISTOLOGICAL CHANGES IN AORTIC TISSUES.DIABETIC GROUPS: The histologic examination of aortic fragments with O-Rd staining from diabetic hamsters revealed morphologic changes specific to diabetic macroangiopathy (Fig. 1B). Intimal thickening, endothelial discontinuities, lipid drops in the subintimal space, and alterations to the middle layer of the arterial wall were observed. The addition of flaxseed and/or vitamin E to the high-fat diet in diabetic hamsters improved the histological aspects of aortic atherosclerosis described in the diabetic hamsters fed an unsupplemented diet. The aortal tissue in the supplemented groups was characterized by slightly swollen endothelium, with moderate intimal thickening and moderate lipid infiltration of the intima (Fig. 2). BODY.DISCUSSION: In DM, oxidative stress occurs, primarily due to the increased production of oxygen free radicals and/or a reduction in the antioxidant defense. Numerous studies have demonstrated that an increase in oxidative stress is implicated in the pathogenesis and progression of diabetic vascular lesions (1,4,6). In the present study, the antioxidative and antiatherosclerotic effects of supplementation with flaxseed (15 g Linum usitatissimum/100 g food), α-tocopherol (40 mg α-tocopherol/100 g food), and their combination were investigated in an animal model that replicates human atherosclerotic lesions. Golden Syrian hamsters appear more predisposed to develop atherosclerotic lesions than rats (30), and present many similarities with the lipoprotein metabolism in humans (31). In hamsters, a 15% flaxseed-supplemented diet is similar in energetic load to the 40 g/day dosage used in human trials. In humans, high doses of 40–50 g flaxseed/day, which corresponds to ~10% of total energy intake have been used in trials (32). In the present study, it was hypothesized that adding vitamin E to the flaxseed diet would lead to improved results for oxidative stress and the prevention of atherosclerotic lesions as there is evidence that the consumption of flaxseed reduces vitamin E (19), and only vitamin E supplementation is able to restore the plasma concentration (20). The present study demonstrated that all three diets (flaxseed, α-tocopherol or their combination) had an antiatherogenic effect in diabetic hamsters, suggested by the prevention of histological lesions (reduced intimal lipid infiltration and discrete alterations to the middle layer of the arterial wall) and by the decreased aortic cholesterol content in supplemented diabetic hamsters compared with those in the unsupplemented diabetic group. The antiatherogenic mechanism of the diets used in our experiment may be associated with changes in metabolic parameters, particularly in glucose concentrations and the lipid profile, and oxidative stress. Flaxseed, α-tocopherol, and the combination of flaxseed and α-tocopherol added to the high-fat diet resulted in similar reductions in serum lipid peroxidation in the diabetic hamsters. Liver lipid peroxidation was significantly diminished by supplementation of the diet by flaxseed alone or in combination with α-tocopherol, but not by α-tocopherol supplementation alone. Moreover, all three diets increased the liver GSH content and the highest concentrations were obtained with the combined diet. These results suggest that the higher antioxidant effects of flaxseed plus vitamin E are due to their synergic antioxidant actions. Flaxseed is the richest source of the lignan SDG. SDG isolated from flaxseed has oxygen radical-scavenging properties that have been demonstrated in vitro by direct hydroxyl radical-scavenging activity (18) or by inhibition of lipid peroxidation (16). Vitamin E is known as a major antioxidant in the cellular antioxidant defense system, which acts by interrupting the propagation of free radical chain reactions. The effect of vitamin E against the prooxidant status associated with DM has been demonstrated by the suppression of diabetes-induced increases in ROS and diminishment of lipid peroxidation (33,34). The synergistic effect of flaxseed oil and vitamin E on oxidative stress has also been observed by Deng et al (35) in male Wistar rats fed a high-fat diet. Similar to the findings of previous studies (36), the diabetic hamsters had higher serum concentrations of serum glucose, TC, TG and non-HDL-C, and lower HDL-C concentrations compared with the control group. The addition of flaxseed, α-tocopherol or their combination to the high-fat diet reduced serum concentrations of glucose, TC and non-HDL in diabetic male hamsters (37,38). The hypocholesterolemic effects of flaxseed may be attributed to ALA, lignans (SDG) or the fiber content (39). In agreement with other studies (40), vitamin E significantly reduced serum TG levels in diabetic hamsters fed an atherogenic diet. In the present study, the addition of vitamin E alone or in combination with flaxseed to the high-fat diet resulted in lower serum concentrations of TC, non-HDL and TG but failed to correct the HDL-C concentrations in diabetic hamsters. Another study has reported that in hamsters with experimental STZ-induced DM fed high a saturated fat and cholesterol diet, supplementation with α-tocopherol increased TC and did not change HDL-C (40). The results of the present study suggest that the antiatherogenic effect of flaxseed, α-tocopherol and their combination added to a high-fat diet in diabetic hamsters is based primarily on the antioxidative role of these components. The improvement of serum glucose and non-HDL were evident and could contribute to the prevention of diabetic macroangiopathy. The antioxidant effect of flaxseed was similar to that of α-tocopherol in diabetic hamsters fed a high-fat diet and the combined diet (Flax + E) did not seem to confer more benefits than flaxseed alone. Moreover, the high dose of ground flaxseed alone may have a better cardioprotective effect than α-tocopherol in diabetic hamsters due to the reduction of TC and non-HDL levels and increase in HDL-C levels. Oxidative stress has been proposed as a pathogenic mechanism of diabetic macrovascular complications, and dietary strategies that ameliorate oxidative stress may represent a promising approach to the prevention of atherosclerosis. The flaxseed-enriched diet may regulate the generation of reactive oxygen species and the metabolism of glucose and lipids, and could consequently play a role in the prevention of major cardiovascular complications in diabetic states. Ground flaxseed combined with α-tocopherol does not appear to confer a clear advantage compared with flaxseed alone in the reduction of oxidative stress and vascular lesion formation in experimental diabetes.

TITLE: Attenuating effect of ABSTRACT.BACKGROUND/OBJECTIVES: Lactobacillus brevis G101 suppresses the absorption of monosodium glutamate (MSG) from the intestine into the blood in mice. Therefore, the attenuating effect of orally administered G101 on monosodium glutamate (MSG) symptom complex was investigated in humans. ABSTRACT.MATERIALS/METHODS: Capsules (300 mg) containing Lactobacillus brevis G101 (1×1010 CFU/individual) or maltodextrin (placebo) was orally administered in 30 respondents with self-recognized monosodium glutamate (MSG) symptom complex for 5 days and the rice with black soybean sauce containing 6 g MSG (RBSM) was ingested 30 min after the final administration. Thereafter, the MSG symptom complex (rated on a 5-point scale: 1, none; 5, strong) was investigated in a double blind placebo controlled study. The intensity of the MSG symptom complex was significantly reduced in respondents of the G101 intake group (2.87 ± 0.73) compared to that in those treated with the placebo (3.63 ± 1.03) (P = 0.0016). Respondents in the placebo group exhibited more of the various major conditions of the MSG symptom complex than in the G101 intake group. Although there was no significant difference in the appearance time of the MSG symptom complex between subjects orally administered G101 and those administered the placebo, its disappearance in < 3 h was observed in 69.9% of subjects in the G101 treatment group and in 38.0% of subjects in the placebo group (P = 0.0841). ABSTRACT.CONCLUSIONS: Oral administration of Lactobacillus brevis G101 may be able to reduce the intensity of the MSG symptom complex. BODY.INTRODUCTION: Monosodium glutamate (MSG), the sodium salt of glutamate, is used worldwide as a flavor enhancer in various processed foods [123]. MSG was extracted from seafood and has been as the source of the umami taste [4]. The Food and Drug Administration (FDA) has classified MSG as a generally recognized as safe (GRAS) substance and no limitation is placed on its use as a food additive. Nevertheless, Kwok first described a complex of symptoms, including numbness at the back of the neck and arms, weakness, and palpitation, after ingestion of a Chinese meal and suggested that MSG was one of the possible causes [5]. These symptoms were referred to collectively as Chinese Restaurant Syndrome (CRS). In 1995, the Federation of American Societies for Experimental Biology (FASEB) proposed the term MSG symptom complex to denote the reactions that occur following ingestion of MSG [6]. When > 5 g may be ingested, it is characterized by unpleasant reactions such as numbness, tingling, headaches, muscle tightness, general weakness, and flushing [7]. Lactic acid bacteria are found in yogurt, cheese, kimchi, and in the gut microbiota [8]. Upon ingestion, lactic acid bacteria confer beneficial effects on the health of the host [9]. Of these lactic acid bacteria, some convert MSG to gamma-aminobutyric acid (GABA), which has been reported to possess various physiological functions that act via the decarboxylation of glutamic acid by glutamate decarboxylase [1011]. Therefore, lactic acid bacteria have been used as initiators to produce GABA-rich foods [12]. We previously isolated Lactobacillus brevis G-101, a kimchi-derived lactic acid bacterium that potently converted MSG to GABA [13]. Lactobacillus brevis G101 reduces the concentration of MSG in the blood of mice fed oral administration in mice: G101 suppresses the absorption of MSG from the intestine into the blood rather than mediating the biotransformation of MSG to GABA [14]. In the present study, to investigate the ability of orally administered Lactobacillus brevis G101 to attenuate the MSG symptom complex, we orally administered G101 or a placebo (maltodextrin) to respondents presenting with self-recognized MSG symptom complex, following ingestion of rice with black soybean sauce (Jajangbap) containing 6 g MSG (RBSM), and then investigated the intensity of the MSG complex in them. This experiment was performed as a double-blind, placebo-controlled study. BODY.SUBJECTS AND METHODS.SUBJECTS: The study subjects consisted of 30 healthy Korean individuals (average age, 23.57 ± 2.36 yrs; 10 male subjects, 23.00 ± 3.46 yrs; 20 female subjects, 23.85 ± 1.60 yrs) who self-identified as MSG-sensitive. Exclusion criteria included consumption of any current medication. The recruitment of subjects was approved by the Committee for the Care and Use of Clinical Study at Kyung Hee University (KHSIRB-14-004) and performed according to CONSORT 2010 guidelines. BODY.SUBJECTS AND METHODS.EXPERIMENTAL PLAN: All 30 subjects participated in an orientation session for this study. The subjects were evenly divided into 2 groups (A and B). In the first week, group A subjects were orally administered one capsule (Lactobacillus brevis G101) at 11:00 am for 5 days and group B subjects took the placebo capsule every day (Table 1). RBSM was ingested 30 min after the final administration of G101, and the intensity and variety of the MSG symptom complex over 6 h were described. In the second week, group A switched with group B and the test was performed for 5 days. The intensity of the MSG symptom complex was rated according to a 5-point scale (1 = none, 5 = strong). Capsules (300 mg) containing 1 × 1010 CFU Lactobacillus brevis G101 or maltodextrin (placebo) were prepared by CTCBIO Inc. RBSM was prepared by mixing steamed rice (180 g) and MSG-contained black soybean sauce. The ingredients of the MSG-contained black soybean sauce (one serving) were as follows: 40 g pork, 40 g onion, 40 g zucchini, 30 g carrot, 50 g cabbage, 1 Tsp black soybean sauce without MSG, 1/2 Tsp soybean oil, 6 g MSG, 1 tsp starch, and 1 2/3 Tsp water. MSG was purchased from a local market. BODY.SUBJECTS AND METHODS.STATISTICAL ANALYSIS: The results obtained through this experiment were analyzed using the statistical analysis system (SAS) 9.3 program. The significance of each sample was established using χ2-test and t-test, as appropriate. BODY.RESULTS.EFFECT OF LACTOBACILLUS BREVIS G101 ON THE INTENSITY OF RBSM-INDUCED MSG SYMPTOM COMPLEX: To evaluate the attenuating effect of Lactobacillus brevis G101 against the MSG symptom complex, we orally administered G101 to volunteers who then ingested RBSM, and after which we evaluated the MSG symptom complex for 6 h in a double-blind, placebo-controlled study (Table 2). The intensity of MSG symptom complex was measured using a 5-point scale (1 = none, 5 = strong). Oral administration of G101 significantly attenuated the intensity of the MSG symptom complex (P = 0.0016). When treated with the placebo, 43.3% of respondents presented with more than a slightly strong MSG symptom complex, but when treated with G101, only 20.0% of respondents exhibited more than a slightly strong response. A significant difference was observed among female subjects, but not among male subjects, indicating that female respondents were more sensitive to the MSG symptom complex than male (Table 3). BODY.RESULTS.EFFECT OF LACTOBACILLUS BREVIS G101 ON RBSS-INDUCED MSG SYMPTOM VARIETY: The effects of orally administered G101 on RBSM-induced self-recognized MSG symptoms (variety) experienced by the respondents were investigated (Table 4). After ingestion of RBSM, the most frequently observed self-recognized symptoms associated with the MSG symptom complex were thirstiness (81.7%) and drowsiness (70.0%), followed by weakness (26.7%), tightness (11.7%), headache (10.0%), nausea (10.0%), dizziness (8.3%), palpitation (1.7%), flushing (1.7%), and indigestion (1.7%). No significant difference was observed between the varieties of self-recognized symptoms experienced by respondents who were orally administered G101 and those administered the placebo. BODY.RESULTS.EFFECT OF LACTOBACILLUS BREVIS G101 ON THE APPEARANCE AND DISAPPEARANCE TIME OF RBSS-INDUCED MSG SYMPTOM COMPLEX: The effect of orally administered G101 on the time at which the RBSM-induced MSG symptom complex appeared was also evaluated (Table 5). There was no significant difference in the appearance time of the MSG symptom complex observed between subjects orally administered G101 and those administered the placebo. When subjects were treated with G101, the appearance for the MSG symptoms complex was less than 1 h for 70.0% of the respondents in the group, and in subjects treated with the placebo, the appearance time was for 76.6% of the group. Next, we investigated the effect of orally administered G101 on the disappearance time for the RBSM-induced MSG symptoms complex (Table 6). There was no significant difference in the appearance time for the MSG symptom complex between subjects orally administered G101 and those administered the placebo. Disappearance of the MSG symptom complex in less than 3 h was observed in 69.9% of subjects in the G101 treatment group and in 38.0% of subjects in the placebo group (P = 0.0841). BODY.DISCUSSION: MSG is frequently used in various canned foods and in food from Chinese restaurant. MSG has been associated with various side effects, including numbness, headaches, migraines, palpitations, tightness, weakness, aching, flushing, sweating, fasciculation, lacrimation, syncope, dizziness, shudder attacks, paresthesia, arrhythmia and tachycardia [1]. Although the oral threshold range for minimal MSG-induced symptoms in humans is considerably variable, in the previous study, the oral threshold range for minimal MSG-induced symptoms in 36 subjects was found to be 1.5 to 12 g, and increased intensity of the MSG symptom response was related to an increase in the oral dose [15]. In this study, we found that oral administration of RBSM, which is containing 6 g of MSG, showed various side effects including thirstiness (56.7%), drowsiness (23.3%), weakness (6.7%), indigestion (6.7%), headache (3.3%), and nausea (3.3%). It was previously shown that the elimination of all food sources containing MSG resulted in a decreased frequency of headaches [3]. These results suggest that the side effects of MSG are dependent on the daily intake amount of MSG, and the oral intake of more than 6 g of MSG may cause its side effect. Therefore, Xiong et al. suggested possible ways to reduce the side effects of MSG, such as taking vitamin C or pre-exposure to a low dose of MSG that may either prevent or reduce the side effects of MSG [3]. Nevertheless, it is not sufficient to reduce only the side effects of MSG. Therefore, we investigated the ability of Lactobacillus brevis G101 to reduce the side effects of MSG. Oral administration of Lactobacillus brevis G101 significantly attenuated the MSG symptom complex. Although G101 did not reduce the variety of side effects, it shortened the disappearance time of the MSG symptom complex. In conclusion, this study suggests that orally administration of Lactobacillus brevis G101 may be able to reduce the intensity of the MSG symptom complex and shorten its disappearance time.

TITLE: Laser Peripheral Iridotomy versus Trabeculectomy as an Initial Treatment for Primary Angle-Closure Glaucoma ABSTRACT.PURPOSE: To compare laser peripheral iridotomy (LPI) with trabeculectomy as an initial treatment for primary angle-closure glaucoma (PACG) with peripheral anterior synechiae (PAS) ≥ 6 clock hours. ABSTRACT.METHODS: Patients were drawn from two randomized controlled trials. 38 eyes of 38 patients (PAS ≥ 6 clock hours) were treated with LPI (group 1) while 111 eyes of 111 PACG patients (PAS ≥ 6 clock hours) underwent primary trabeculectomy (group 2). All patients underwent a comprehensive ophthalmic examination at baseline and at postoperative visits and were followed up for a minimum of one year. ABSTRACT.RESULTS: Group 2 had higher baseline IOP (45.7 ± 14.8 mmHg versus 34.3 ± 14.3 mmHg) than group 1 and more clock hours of PAS (10.4 ± 1.9 versus 9.0 ± 2.2). IOPs at all postoperative visits were significantly lower in group 2 than in group 1 (p = 0.000). Five eyes in group 1 required trabeculectomy. 17 of the 38 eyes in group 1 (44.7%) required IOP-lowering medications as compared to seven of the 111 eyes in group 2 (6.3%). Cataract progression was documented in 2 eyes (5.3%) in group 1 and 16 eyes (14.4%) in group 2. ABSTRACT.CONCLUSIONS: Primary trabeculectomy for PACG (PAS ≥ 6 clock hours) is more effective than LPI in lowering IOP. BODY.1. INTRODUCTION: Primary angle-closure glaucoma (PACG) is the major type of primary glaucoma in China [1–4]. The management of PACG in China is different from the recommendations of the American Academy of Ophthalmology Preferred Practice Patterns (PPP) and other published guidelines [5–7]. For primary angle closure (PAC) and PACG, the PPP recommends laser peripheral iridotomy (LPI) to eliminate pupillary block followed by a treatment strategy "similar to that for POAG" [8]. Accordingly, LPI is used as the first-line treatment for all patients with PAC or PACG, medication is added as needed, and surgery is considered when the intraocular pressure (IOP) cannot be controlled with maximum tolerated medications. In China, however, trabeculectomy is considered a primary option for PACG and is generally undertaken if peripheral anterior synechiae (PAS) are greater than 6 clock hours [9, 10]. To the best of our knowledge, there is no comparative study of LPI versus primary trabeculectomy in PACG to support such an approach. Using data from patients enrolled in two separate randomized controlled trials [11, 12], we compared the IOP-lowering efficacy and safety of LPI versus trabeculectomy as an initial treatment for PACG with PAS ≥ 6 clock hours. BODY.2. METHODS: Patients included in this study had participated in 2 randomized clinical trials (RCTs) for PACG. Both trials were conducted in accordance with the tenets of the Declaration of Helsinki and approved by the ethics committee of the Tongren Eye Centre, Capital Medical University. Written informed consent was obtained from all subjects for participation in the original trials. Patients undergoing primary trabeculectomy for PACG had participated in a multicenter RCT (registration number: ChiCTR-TCR-00000218) [12]. This RCT was conducted in four clinical collaborative centers of Beijing Tongren Hospital: Handan 3rd Hospital (Hebei Province, China), Anyang Eye Hospital (Henan Province, China), Fushun Eye Hospital (Liaoning Province, China), and the Chenzhou Eye and Optometry Center (Hunan Province, China). PACG was defined as primary angle closure with glaucomatous optic neuropathy and corresponding visual field defects, and patients were recruited from the four centers between April 2006 and November 2007. The primary purpose of the trial was to report the efficacy and complications of trabeculectomy with or without releasable sutures in PACG. Patients who underwent LPI as an initial treatment were part of another RCT (registration number: ChiCTR-TRC-00000034, http://www.chictr.org.cn) conducted at the Handan 3rd Hospital [11]. The purpose of this RCT was to investigate the role of laser iridotomy (with or without iridoplasty) in patients with synechial PAC or PACG. The definition of PACG was the same as that used in the trial mentioned above; consecutive cases of PAC and PACG presenting to the hospital between October 1, 2005, and October 31, 2006, were recruited for this trial. BODY.2. METHODS.2.1. PATIENT SELECTION: The inclusion criteria for the current study were as follows: PACG: defined as primary angle closure with glaucomatous optic neuropathy and visual field defects [13]Age 40 years or morePAS ≥ 6 clock hoursMinimum follow-up of one year. Patients with acute angle-closure glaucoma were excluded. As part of the original clinical trial, all patients had undergone a comprehensive ophthalmic examination including refraction, Goldmann applanation tonometry, static and dynamic gonioscopy (manipulation) using a one-mirror Goldmann gonioscope [14, 15], slit-lamp examination, fundus examination, and automated perimetry (Humphrey Field Analyzer 750i, Carl Zeiss Meditec; Sita Fast strategy; and 24–2 threshold test). These examinations were performed at baseline and at postoperative visits scheduled at month 1, month 3, month 6, month 12, and month 18. Postoperative visits were scheduled on day 1, day 3, week 1, week 2, month 1, month 3, month 6, month 12, and month 18 following the LPI or trabeculectomy. BODY.2. METHODS.2.2. LASER PERIPHERAL IRIDOTOMY: All laser procedures were performed by one of two senior glaucoma specialists. 2% pilocarpine was applied, and iridotomy was performed under topical anesthesia using a Nd:YAG laser (YL-1600; NIDEK Co. Ltd., Japan) using an Abraham contact lens (Ocular Instruments Inc., Bellevue, USA). A treatment site was selected in the superior nasal iris or in a crypt where present. The treatment was initiated with a single 4 mJ pulse, the power was adjusted, and the treatment was continued to obtain a 0.2 mm opening; patency was determined by direct visualization of the posterior chamber. In accordance with local practice, IOP-lowering medication was initiated if the IOP was greater than 21 mmHg following laser and confirmed by a repeat reading on the same day [11]. BODY.2. METHODS.2.3. TRABECULECTOMY: Surgery was performed under topical or peribulbar anesthesia using a standard surgical technique. The eye was prepared using a standard aseptic technique and draped to isolate the lashes. A lid speculum was inserted and a 7/0 superior rectus muscle traction suture was placed. A limbus-based conjunctival flap was created using a 10 mm incision through the conjunctiva and Tenon's capsule approximately 8–10 mm from the limbus. The flap was dissected forwards and hemostasis achieved with monopolar diathermy. A half-thickness 4 × 3 mm2 rectangular scleral flap was fashioned, and cellulose sponges soaked in MMC (0.3 mg/ml) were applied under the scleral flap, conjunctiva, and Tenon's capsule for a duration determined by the surgeon based on an assessment of risk factors. Irrigation with balanced salt solution was performed to wash out residual MMC solution. A paracentesis was created, a 2 × 1.5 mm trabeculectomy block excised, and an iridectomy performed. The scleral flap was sutured with 10-0 monofilament, BSS was injected into the anterior chamber to assess flow, and the conjunctiva was closed with a single running 8/0 vicryl suture [12]. Visual acuity was recorded with a decimal chart and converted to the logarithm of minimum angle of resolution (LogMAR) format. Finger counting, hand movement, and light perception were recorded as 1.5, 2.0, and 2.5 on the LogMAR scale. At each postoperative visit, a trained technician measured the IOP twice using an applanation tonometer and recorded the average. If the difference between the two measurements was more than 2 mmHg, a third measurement was performed, and the average of the two closer results was recorded. All IOP measurements were performed between 8 am and 12 am. At each site, trained observers used standard LOCS III photographs to assess the lens. Cataract progression was defined as an increase of 2 or more units greater than baseline in any LOCS III category (nuclear, cortical, or posterior capsular opacity). Progression was analyzed in all eyes with a minimum follow-up of 12 months; for those whose longer follow-up was available, it was determined at the 18-month visit. The shallow anterior chamber (AC) was categorized as grade I = peripheral iris-cornea touch; grade II = midiris-cornea touch; and grade III = central cornea-lens touch [16]. Hypotony was defined as IOP ≤ 5 mmHg [17]. At each visit, other complications and interventions, if undertaken, were recorded. BODY.2. METHODS.2.4. STATISTICAL ANALYSIS: One eye of each patient was randomly selected for the analysis. Statistical analysis was carried out using SPSS 15.0 (SPSS Inc., Chicago, USA). Independent t-test was used to compare the difference between the groups, and the chi-square test was used to compare the difference in IOP-lowing medication and cataract progression between the groups. A p value < 0.05 was considered significant. BODY.3. RESULTS: 38 eyes of the 38 patients with PACG that underwent primary iridotomy comprised group 1 while group 2 consisted of 111 eyes of the 111 PACG patients who had undergone trabeculectomy. The baseline characteristics of the two groups are summarized in Table 1. Patients in group 2 were slightly younger and had higher baseline IOP and more clock hours of PAS. Table 2 shows the postoperative IOP and visual acuity. At each visit, the IOP was significantly lower in group 2 (p < 0.001). Five eyes in group 1 required trabeculectomy: 1 eye at 6 months, 2 eyes at 12 months, and 2 eyes at 18 months. 17 of the 38 eyes (44.7%, CI: 28.9%–60.5%) in group 1 required a mean of 1.8 IOP-lowering medications while seven of the 111 eyes (6.3%, CI: 1.8%–10.8%) in group 2 required a mean of 1.1 medications (Pearson's chi-square value = 30.940, p < 0.001). 19 eyes in group 2 developed a transient shallow anterior chamber (AC), but there was no instance of lens-cornea touch. 16 eyes (including 13 eyes with shallow AC) experienced transient hypotony. Hypotony lasted one day in 14 eyes and one week in 2 eyes and all recovered spontaneously. One eye developed hypotony maculopathy at one month after surgery that was resolved after IOP increased 3 weeks later. In one eye, the IOP increased to 60 mmHg at 3 months postsurgery and required cyclophotocoagulation. Best-corrected visual acuity at 18 months was 0.5 ± 0.5 in group 1 and 0.6 ± 0.7 in group 2. Four eyes (10.5%) in group 1 lost one line of vision, 3 eyes (7.9%) lost 2 lines, and 3 (7.9%) lost more than 2 lines. In group 2, thirteen eyes (11.7%) lost 1 line, 9 eyes (8.1%) lost 2 lines, and 11 eyes (9.9%) lost more than 2 lines. Six eyes in group 1 and 20 eyes in group 2 lost ≥2 lines (Pearson's chi-square value 0.098, p = 0.755). The absolute risk for ≥2 lost lines of vision in the trabeculectomy group was 18% (20 of 111) compared to 16% (6 of 38) in the LPI group. The absolute risk increased with trabeculectomy is 2% and the number needed to harm (NNH) is 50. Cataract progression as defined was documented in 2 eyes (5.3%, CI: −1.8%–12.4%) in group 1 and 16 eyes (14.4%, CI: 7.9%–20.9%) in group 2 (Pearson's chi-square value = 2.232, p = 0.135). The absolute risk increased in cataract formation with trabeculectomy is 9% and translates into a NNH of 11. BODY.4. DISCUSSION: The PACG treatment strategy formulated by the glaucoma group of the Chinese Ophthalmology Society is different from that formulated by other published guidelines. As per Chinese guidelines, LPI is suggested for "patients with PAS < 180° without optic disc and visual field damage" (an indication endorsed without data by a recent publication), while primary trabeculectomy is recommended for PACG with PAS ≥ 180° [9, 18]. A 2005 survey of clinical practice in PACG diagnosis and treatment found that 73% of Chinese glaucoma specialists preferred trabeculectomy as the initial treatment for PACG with PAS ≥ 180° [10]. Evidence for such an approach is however lacking, and to the best of our knowledge, our study is the first study to compare primary LPI with primary trabeculectomy for PACG. LPI is considered a noninvasive, simple, and safe intervention in PACS, PAC, and PACG [19]. The effect of a LPI would depend on the amount of angle that is available for filtration, and this can be estimated clinically by the extent of PAS [18, 20]. IOP control following LPI as reported in the literature varies considerably, and the therapeutic effect seems to decrease over time [21, 22]. In a retrospective analysis of 131 cases (251 eyes) in China, 18 eyes with PACS, 98 eyes with PAC, and 129 eyes with PACG that underwent LPI were followed up for 9.2 ± 3.7 years [23]. Eyes with PAC and PACG had PAS < 6 clock hours. At the final follow up, 16/18 PACS, 38/98 PAC, and 814/129 of PACG eyes were controlled (defined as IOP less than 21 mmHg) without medications. 60% of cases required additional medications and 13% needed filtering surgery. A study from India evaluated the long-term outcome of PACG following laser iridotomy [24]. 70 consecutive patients with PACG whose IOP remained >21 mmHg despite a patent iridotomy had their IOP controlled by medications or trabeculectomy and were followed up over a 6-year period. A trabeculectomy without antimetabolites was performed if IOP was >21 mmHg on maximal tolerable medical therapy, if there was evidence of progression of the disc/visual field, or if the patient was noncompliant. 46 (65.7%) eyes were controlled medically with 26 (57%) eyes requiring two topical medications. 24 (34.3%) eyes required trabeculectomy at various times during follow-up. The extent of PAS was not reported in this study, but reports suggest that the therapeutic effect of LPI decreases over time and many cases need filtering surgery. The results seem consistent with the biologically plausible hypothesis that LPI is more successful in those with relatively undamaged trabecular meshwork [18]. The literature seems to suggest that, despite the presence of a patent LPI, eyes with established PACG and a certain (currently undefined) extent of PAS require further treatment to control IOP and that medical therapy fails in a significant number of cases necessitating filtering surgery [18, 25]. In the current investigation, the IOP during follow-up was significantly lower in the trabeculectomy group. Furthermore, a larger number of patients who underwent LPI needed medical treatment as compared to those who underwent trabeculectomy. In this developing country, setting patients may not be able to afford medical treatment or are generally not adherent or persistent with it. To simulate a real-world condition, we did not provide free IOP-lowering medication and found that 24% of patients could not afford long-term medication. Moreover, 5 eyes (13.2%) of patients who underwent LPI with baseline PAS ≥ 6 clock hours (group 1) had to undergo trabeculectomy during the follow-up period. The poor effectiveness of IOP lowering following LPI for PACG with ≥6 hours of PAS would seem important in selecting treatment within the well-known constraints of a developing country, including a possible one shot at treatment. We therefore suggest that 6 clock hours of PAS can be considered a provisional clinical threshold for LPI in PACG, as this is likely to change with further research and will be different for individual cases. Our findings lend credence to a recently published algorithm for the management of angle-closure disease that recommended a ≥6 clock hour threshold for PAS; that algorithm was based mainly on biological plausibility [18]. Considering the problems of medical treatment in developing countries, a policy of primary trabeculectomy for PACG with ≥6 clock hours of PAS might merit consideration. In fact, even eyes with <6 clock hours of PAS may be considered for a trabeculectomy if the socioeconomic situation so dictates [23]. The choice of initial treatment must also consider the issue of complications. Consistent with most other publications, we did not encounter any significant complications in the LPI group [11]. While complications with trabeculectomy are more frequent, the incidence of severe sight-threatening complications is now less than what it was 20 years ago; we encountered mainly transient shallow chamber and hypotony. It must be kept in mind that in our study, trabeculectomy was undertaken by glaucoma specialists in the context of a clinical trial and the potential for sight-threatening complications remains. Importantly, even in the context of a clinical trial, 18% of eyes undergoing trabeculectomy lost 2 or more lines of vision. However, the number needed to harm for ≥2 lost lines of vision is an acceptable 50. Incision-induced astigmatism as well as progression of cataract was the major reason for visual loss, but both would be considered treatable. 19% of patients in undergoing trabeculectomy had astigmatism of >1D at 6-month visit, and 14.4% had obvious progression of lens opacity. The NNH for progression of cataract with trabeculectomy was 11. While successful intervention is available for cataracts, this low NNH that could lead to an increase in the burden of cataract is a disadvantage with trabeculectomy. The choice for the initial intervention for PACG in China and other developing countries must weigh up effectiveness versus complications as well as the need for additional medications and further interventions. While LPI is safe and easy, there is a more need for intensive medications and surgical intervention. The role of cataract extraction in primary angle-closure disease is evolving but is again more likely to be useful for those with angles less compromised by PAS [18, 26]. Trabeculectomy is better than LPI for lowering the IOP, but it does have the potential for more loss of visual acuity and for serious complications. Accordingly, it is probably best to reserve primary surgical intervention for established PACG with ≥6 clock hours as these are unlikely to do well with laser alone. While not statistically significant in this study, the higher chances for progression of cataract following trabeculectomy raise the question of combining filtration with cataract surgery, an issue not addressed in this study. In addition to clinical factors, any decision will also depend on the availability of lasers and surgeons and costs. One of the limitations in our study is the small sample size in group 1 and the relatively short follow-up time. As few patients progressed during this time, we could not analyze the relationship between IOP and the visual field progression, but IOP would usually be considered an acceptable surrogate. Also, our results are derived from patients enrolled in RCTs, and the effectiveness in the real world is likely to be different. Furthermore, as it was not part of this study, we cannot comment on the increasingly popular role of phacoemulsification or its combination with trabeculectomy in the management of PACG [18]. The low NNH for cataracts with trabeculectomy could however be used as an argument for combining the two surgical procedures. Finally, while the definition of PACG used in both trials was the same, patients for this study were drawn from two separate trials that asked different questions. This could introduce significant bias. The original LPI RCT (registration number: ChiCTR-TRC-00000034, http://www.chictr.org.cn) [11] that randomized patients into two different treatment groups (iridotomy or iridotomy plus iridoplasty) found no significant difference in IOP, medications, need for surgery, or visual function between groups at the 1-year visit. This RCT showed that in eyes with PACG, both iridotomy alone and iridotomy combined with iridoplasty provide a significant and similar reduction in IOP. For the trabeculectomy RCT (registration number: ChiCTR-TCR-00000218) [12], patients were randomly allocated to permanent or releasable sutures. This study too did not demonstrate any significant differences between the groups. Accordingly, we felt that analyzing patients from either arm of the two trials for the current study was acceptable but acknowledge that the possibility of unknown differences does exist and could affect interpretation. However, as the baseline IOP was higher and PAS more extensive in the trabeculectomy group, the conclusion in favor of trabeculectomy is likely to stand. In conclusion, our results support the Chinese guidelines for management of PACG. In PACG with ≥6 clock hours of PAS, primary trabeculectomy is more effective than LPI in lowering IOP and significantly requires less medication but with more progression of cataract and more loss visual acuity. In developing countries, such as China, trabeculectomy can be considered a primary option for PACG with this degree of PAS.

TITLE: Changes in proprioception and pain in patients with neck pain after upper thoracic manipulation ABSTRACT: [Purpose] The purpose of this study was to conduct cervical stability training and upper thoracic manipulation for patients with chronic neck pain and then investigate the changes of cervical proprioception and pain. [Subjects and Methods] Subjects were 30 workers with mechanical neck pain, who were randomly divided into an upper thoracic manipulation group and a cervical stability training group. Upper thoracic manipulation after cervical stability training was conducted for the upper thoracic manipulation group, and only stability training was conducted for the cervical stability training group. The intervention period was six weeks, and consisted of three sessions a week, each of which lasted for 30 minutes. For proprioception measurement, an electro-goniometer was used to measure reposition sense before and after the intervention. The visual analogue scale was used to assess pain. [Results] After the intervention, the error angle was significantly smaller in flexion and right left side-bending, and pain was significantly reduced in the upper thoracic manipulation group. According to the post intervention comparison of the two groups, there were significant differences in the proprioception and pain values. [Conclusion] Conducting both cervical stability training and upper thoracic manipulation for patients with chronic neck pain was more helpful for the improvement of proprioception and pain than cervical stability training alone. BODY.INTRODUCTION: Neck pain is a disease that most people experience at least once in their life time. Neck pain is the main cause of an increase in economic and social health costs. To treat neck pain, physical therapists often use therapeutic exercise, traction, joint mobilization, and manipulation1, 2). In the case of the patients with chronic neck pain, because of overuse, repetitive trauma, serious trauma and muscle weakness, the range of elasticity of the non-contractile tissues becomes enlarged, and stablize the neutral position, and contractile tissues become weak. As a result, instability increases in the spinal segments, resulting in failure to maintain the neutral position. Joint instability is accompanied by loss of somatosensory system function. In addition, joint instability causes hyper-mobility of cervical segments, and restricts the movement of the thoracic segments, which ultimately leads to functional restriction2). Manipulation and mobilization are clinically employed often to improve cervical instability and functional movement. Thoracic manipulation and joint mobilization help to increase the restricted mobility of the spinal segments, to improve cervical mobility and stability, and to recover functional movement and proprioception, and ultimately reducing pain and functional disorder3, 4). In order to recover cervical proprioception in patients with chronic neck pain, a therapeutic approach is conducted to improve cervical instability, joint mobility, and neuromuscular control. Cranio-cervical flexor (CCF) training aims at recovery of control of the muscular system around the cervical spine and the recovery of cervical proprioception. For improvement in cervical joint mobility, manipulation and mobilization are performed5). Previous studies have reported that direct cervical manipulation is a dangerous cervical treatment, because it can reduce the function of the spinal artery. For that reason, CCF training is widely employed to improve proprioception, pain and range of motion (ROM), and reduce the neck disability index (NDI) score of patients with neck pain4, 6). Recently, indirect thoracic manipulation and mobilization have frequently been used to treat patients with neck pain, because it is safer than direct manipulation. Spinal manipulation elicits immediate effects on recovery of function, such as pain alleviation and ROM improvement, than conservative physical therapy. In addition, according to previous studies, manipulation is more effective at reducing pain than conservative muscle training7). Nevertheless, there is still a lack of research evidence supporting the efficacy of manipulation. Therefore, the purpose of this study was to conduct cervical stability training (CST) and upper thoracic manipulation (UTM) for patients with chronic neck pain and investigate the changes in cervical proprioception and pain. BODY.SUBJECTS AND METHODS: The subjects were 30 subjects with chronic mechanical neck pain who agreed to participate in this study. The inclusion criteria were: unilateral or bilateral pain in the posterior neck or shoulder region, and pain in the cervical region when moving or palpating the cervical region, which had lasted for more than 12 weeks, and was rated higher than four on a visual analogue scale (VAS). The exclusion criteria were: a diagnosis of cervical radiculopathy, previous experience of spinal manipulation, a history of fracture or dislocation of the cervical or thoracic spine, a history of surgical operation on the cervical or thoracic spine, hypertension, heart disease, or pregnancy. The research protocol was approved by the Research Ethics Committee for Human Research of Konyang University, Korea. UTM comprised 2 methods. Method 1: Upper thoracic lift manipulation. The subjects were asked to lock their fingers together, put their hands on the back of the neck, and sit in a chair without a back support. Then, a rolled towel was placed over the target spinal segment. A therapist stood behind the subject, and leant forward toward the rolled towel, or the target spinal segment. The therapist held the forearm of the subject, and flexed the subject's neck and upper thoracic completely up to the spinal segment, creating a force against the rolled towel using the subject's forearm, and held the subject's elbow in a horizontal adduction motion while lifting the subject toward the therapist in the posterior-anterior direction; and then applied thrust manipulation to the subject along with horizontal adduction and expiration4). Method 2: Thoracic manipulation in the supine position. The therapist stood next to the subject. And placed a hand on the subject's transverse process on the inferior segment of the target spinal segment. The subject puts his/her arms on the chest or locked their fingers at the back of the head and neck. The therapist held the subject's arms to keep the subject close, then put the manipulation hand on the target spinal segment and used the supporting hand to fix the subject's elbows with the therapist's forearm. After performing flexion up to the subject's target segment, the therapist applied thrust manipulation in the anterior-to-posterior direction while the subject inhaled and exhaled. The target segments were T1 to T44). CTS consisted of static stability training for re-education and dynamic stability training for muscle endurance and power8). The static stability training used a pressure biofeedback unit (PBU), and it was based on CCF training for activating the deep muscles such as the longus colli and longus capitis. In dynamic stability training, the subjects maintained cervical flexion, and active assistive and active methods were conducted. After they had maintained cervical stability, the subjects performed dynamic training through extremity movement. The subjects were asked to pay attention to the cervical stability during training9). To measure the cervical proprioception of the patients with chronic neck pain, the cervical reposition test was conducted using an electro-goniometer (Mobee, Bitburg, 2013, Germany). The study subjects were asked to sit in an upright position comfortably in a chair. The target position was randomly chosen from among 30%, 60%, and 90% of the maximal ROM. The subjects kept the target position for 3 seconds, repeated it 5 times10). For measurement, the reposition test was conducted at the set angle 3 times. The rest time between measurements was 15 seconds. Measurement directions were set to flexion, extension, right and left side-bending. The subjects were asked to sit upright and wear black sunglasses to inhibit their vision, and to move actively toward the target position in a random order. The difference between the target angle and the actual angle was used in the analysis11). All subjects were asked to mark on a VAS scale ranging from 0 to 10 for the subjective assessment of pain. VAS is a simple way for the subjects to express their level of pain and has high reproducibility. It is also an evaluation method with high validity (r=0.77) and reliability (r=0.99)12) (Table 1Table 1.Comparison of proprioception and pain within groups and between groups (N=30)Manipulation group (n=15)Control group (n=15)Flexion (angle)Pre6.07 (2.02)5.67 (2.06)Post2.47 (1.55)†**4.27 (1.44)†,‡**ExtensionPre4.87 (1.81)4.87 (2.48)Post3.27 (0.96)†**3.53 (1.81)†**Rt sidebendPre6.20 (1.79)6.73 (2.55)Post1.87 (1.30)†**5.00 (2.14)†,‡**Lt sidebendPre6.60 (1.55)6.53 (1.89)Post1.00 (1.20)†**4.67 (1.88)†,‡**VASPre7.13 (0.83)6.93 (1.44)Post1.93 (0.70)†**3.40 (0.74)†,‡**Values are expressed as mean (SD). *p<0.05, **p<0.01, †Within group comparison, ‡Between group comparison). For data analysis, SPSS version 18.0 (SPSS, Inc., Chicago, USA) was used. Frequency analysis and descriptive statistics were employed to compare the general characteristics of the subjects. The independent t-test was conducted to compare the thoracic manipulation group and the CST group. The paired t-test was performed to compare the pre- and post intervention results of the UTM and the CST. The level of statistical significance was chosen as 0.05. BODY.RESULTS: There were 8 male and 7 female in the manipulation group, and 7 male and 8 female in the control group. The mean age of the subjects was 30.80 years in the manipulation group and 28.07 years in the control group. The average weight, height and BMI were 63.23 kg, 164.87 cm and 23.20 kg/m2, respectively in the manipulation group, and 62.89 kg, 168.13 cm and 21.93 kg/m2, respectively in the control group. The UTM showed significantly smaller repositioning error in flexion and right and left side-bending and greater reduction in pain than the CST group (p<0.01). Both the UTM and CST groups showed significant differences in proprioception and pain after the intervention (p<0.01). BODY.DISCUSSION: In this study, CST was conducted for the patients with chronic neck pain, followed by UTM for six weeks. After that, the changes in proprioception and pain were analyzed. After the interventions, the UTM group showed significant reductions in error angles which were less than those in the CST group, and the pain level of the UTM group was also significantly reduced. Proprioception receptors, such as mechanoreceptors and free nerve endings, are found in the skin, facial, muscles, tendons, joint capsules, and ligaments. When active or passive movement occurs, receptors transmit the movement information to the central nervous system (CNS)13). High velocity low amplitude manipulation stimulates mechanoreceptors located in the muscles, ligaments, and joint capsules improving their responses in transmitting to the CNS; thus, helping the recovery of proprioception. It is known that manipulation mechanically stimulates joint capsules and thus leading to increased mobility of the spinal segments and increases ROM, which lessens the fear of movement by smoothly transmitting movement information to the CNS, thereby reducing pain1, 14). According to many previous studies, the combined approach of a therapeutic exercise program and manipulation is more effective at recovering the proprioception of patients with neck pain and their functions through pain reduction than any other kind of intervention15). Cassidy et al.16) independently conducted both manipulation and joint mobilization for patients with mechanical neck pain, and reported the two groups showed significant improvements in pain and ROM after the interventions. Joint mobilization conducted for the patients with chronic RA helped to increase their pain threshold17). Another hypothesis regarding pain reduction after manipulation is that beta-endorphins are expressed and that this alleviates pain18). According to Vernon et al.19), after manipulation, beta-endorphins increased for five minutes, but 15 minutes later, their level had returned to its original state. According to the results of the present study, proprioception and pain were significantly reduced after UTM, a result that is consistent with the empirical evidence of previous studies. Another proposal is that spine manipulation activates type I (static movement) and type II (dynamic movement) mechanoreceptors, which are distributed in passive and active structures, sending signals through the CNS to the periaqueductal grey area, the pain control system around the cerebral aqueduct of mid-brain, which increases the pain threshold to control pain20). The results of this study show that the thoracic manipulation group displayed more significant differences in pain reduction than the cervical stability group, and that CST also improved proprioception and pain. According to the study of Panjabi21), cervical stability is maintained by the stability passive and active tissues and the capability of neuromuscular control. The purpose of the passive system is to use the elasticity of passive tissues including ligaments, joint capsules, and tendons to stabilize the neutral position. The purpose of the active system is to use the force generated by active tissues like muscles to maintain the neutral position. In addition, the neuromuscular control system controls and amplitude of the overall range and mobility. The CST result was consistent with the theory that training improves the stability the neutral position and its maintenance through the activation of the active tissues of muscles. Despite these results, this study had several limitations. For example, the number of study subjects small, and there was no long-term intervention of manipulation or CST. Therefore, we consider that it will be necessary to increase the number of study subjects and lengthen the intervention period to prove the effects of manipulation. In the present study, the repositioning test was conducted to measure the propriocetive deficit. However, this is an indirect method, not a direct one, of measuring proprioception. For that reason, it is impossible to generalize the results. Therefore, we think that a more objective tool and measurement method will be needed to investigate proprioception. In summary, the UTM group showed significant improvements in cervical proprioception, and significant reduction in pain, which were larger than those of the CST group. The findings of this study indicate that combine stability training and thoracic manipulation for patients with neck pain improves proprioception and reduces pain level.

TITLE: A single consumption of curry improved postprandial endothelial function in healthy male subjects: a randomized, controlled crossover trial ABSTRACT.BACKGROUND: Curry, one of the most popular foods in Japan, contains spices that are rich in potentially antioxidative compounds, such as curcumin and eugenol. Oxidative stress is thought to impair endothelial function associated with atherosclerosis, a leading cause of cardiovascular events. The aim of this study was to determine whether a single consumption of curry meal would improve endothelial function in healthy men. ABSTRACT.METHODS: Fourteen healthy male subjects (BMI 23.7 ± 2.7 kg/m2; age 45 ± 9 years) were given a single serving of curry meal or spice-free control meal (180 g of curry or control and 200 g of cooked rice; approximately 500 kcal in total) in a randomized, controlled crossover design. Before and 1 hr after the consumption, fasting and postprandial flow-mediated vasodilation (FMD) responses and other parameters were measured. ABSTRACT.RESULTS: The consumption of the control meal decreased FMD from 5.8 ± 2.4% to 5.1 ± 2.3% (P = 0.039). On the other hand, the consumption of the curry meal increased FMD from 5.2 ± 2.5% to 6.6 ± 2.0% (P = 0.001), and the postprandial FMD after the curry meal was higher than that after the control meal (P = 0.002). Presence of spices in the curry did not alter significantly the systemic and forearm hemodynamics, or any biochemical parameters including oxidative stress markers measured. ABSTRACT.CONCLUSIONS: These findings suggest that the consumption of curry ameliorates postprandial endothelial function in healthy male subjects and may be beneficial for improving cardiovascular health. ABSTRACT.TRIAL REGISTRATION: UMIN Clinical Trials Registry 000012012. BODY.BACKGROUND: Curry originated in Indian traditional diet, and has become widely eaten throughout the world, especially in Asia [1]. In fact, curry is one of the most popular foods in Japan [1]. Japanese curry is often milder in flavor and thicker in consistency than the traditional Indian curry, and usually is served with cooked rice [1]. With a good amount of meat and vegetables in it, Japanese curry can make a convenient and nutritious meal for people of all ages [1]. While being mild in flavor, Japanese curry still contains an abundant amount of spices, some of which are high in antioxidants. For example, turmeric contains antioxidant yellow pigment, curcumin, which is known to have many health benefits such as vasoprotective, antiinflammatory, anticarcinogenic, and neuroprotective effects [2,3]. Clove contains antioxidant aromatic oil, eugenol, which is also known to have some health benefits such as vasoprotective and pulmonary protective effects [4,5]. Epidemiologic studies have shown that curry improves pulmonary function in Asian elderly adults [6] and curry consumption improved cognitive performance of nondemented elderly Asians [7]. However, few intervention studies about curry have been reported. It is well known that postprandial hyperglycemia is a contributing factor to the development of atherosclerosis and is a risk factor for cardiovascular events. A meta-regression analysis showed that the progressive relationship between glucose levels and cardiovascular risk extends even in subjects with normal glucose tolerance below the diabetic threshold [8]. Although mechanism by which postprandial hyperglycemia induces vascular dysfunction is not fully understood, a review by Mar and Bruno points out that oxidative stress-mediated disruptions in nitric oxide homeostasis have been implicated as key events leading to vascular dysfunction [9]. Glucose loading produced a decrease in endothelial function and an increase in a marker of oxidative stress in normal and diabetic subjects [10,11]. Hyperglycemia in response to oral glucose loading rapidly suppressed endothelium-dependent vasodilation, probably through increased production of oxygen free radicals [11-13]. Accumulating evidence suggests that endothelial dysfunction plays a crucial role in the development and progression of atherosclerosis. The endothelium is suggested to be a target of damage in the postprandial state [14-16]. The aim of this study was to determine whether a single consumption of a dish of Japanese curry and rice would improve postprandial endothelial function in healthy men. BODY.METHODS.SUBJECTS: In December 2012, 18 healthy males aged 33 to 64 years were recruited for the study. We selected healthy male subjects who do not have any history of hypertension, diabetes mellitus, and dyslipidemia, to avoid the possibility of any influence on the endothelial function by these factors as well as by menstrual cycle. The study protocol was approved by Tana Orthopedic Surgery Institutional Review Board. Informed consent for participation in the study was obtained from all subjects. BODY.METHODS.STUDY DESIGN: The study was performed at House Foods Corporation during the period from January 2013 to February 2013. Subjects were randomized into a control-first group and a curry-first group. Each subject was given a single serving of test meal (control meal or curry meal) in a crossover manner with more than one week in between the two test meals. Fasting and postprandial measurements were taken by the primary investigator under blinded condition as follows. Subjects were asked not to take any nutritional supplements on the day before the test and were fasted for at least 12 hours overnight prior to the fasting measurements. No other instructions were given to the subjects. After the fasting measurements, subjects were given 180 g of either control (198 kcal) or curry (187 kcal) and 200 g of cooked rice (294 kcal) by a support staff, and one hour afterwards, postprandial measurements were taken. A preliminary test which we had made before this study showed that a measurement one hour after the intake of the control meal showed low endothelial function in healthy subjects but this effect was not observed two hours after the intake of the control meal. For this reason, we thought that the single point measurement one hour after meal was appropriate for studying curry effects on postprandial endothelial dysfunction in healthy subjects. The curry consisted of ground beef, tomato, tomato puree, some seasonings, and a blend of spices (Table 1). The control was prepared as the curry except that all the spices were removed from the formula; it tasted like a meat sauce for spaghetti. The nutritional compositions of the control meal and curry meal both with 200 g of cooked rice were essentially the same as shown in Table 2. Table 1 Composition of the spice blend used in a single serving portion of the curry meal Spice Amount Clove (g) 0.9 Coriander (g) 1.8 Cumin (g) 0.9 Garlic (g) 3.6 Ginger (g) 2.7 Onion (sautéed) (g) 9 Red pepper (g) 0.09 Turmeric (g) 4.5 Table 2 Composition of a single serving portion of the test meal with 200 g of rice Component Control Curry Energy (kcal) a 492 481 Protein (g) b 15 16 Fat (g) c 11 10 Carbohydrate (g) d 82 82 Dietary fiber (g) e 3 3 Sodium (g) f 1 1 a Modified Atwater methods. b Kjeldahl nitrogen determination method (conversion factor: 6.25). c Ether extraction method. d Carbohydrate was calculated from protein, fat, ash (direct ashing), and moisture (oven drying at 105°C for 16 hours). e Modified Prosky method. f Inductively coupled plasma emission spectrometry. Single serving portions (180 g) of both the curry and the control were packed and sterilized in retort pouches and were stored at room temperature until use. They were heated in boiling water for five minutes just before serving with 200 g of cooked rice. BODY.METHODS.FMD AND HEMODYNAMIC MEASUREMENTS: All measurements were taken in the morning, in a quiet, air-conditioned room maintained at 24°C and 50% relative humidity. The subjects rested quietly in the supine position for 20 min before and during each FMD measurement. Just before the cuff inflation, systolic and diastolic blood pressure was measured on the left arm. FMD was measured on the right arm as described previously [17] with a UNEXEF18G ultrasound imaging system programmed specifically for FMD measurements with the autocuff (UP310), the semi-automatic vessel tracking (X-link), and the flow mode (uFFLOW) options (UNEX Co., Nagoya, Japan). After the brachial artery diameter was determined for the baseline arterial diameter from a longitudinal image of the artery acquired 5 to 10 cm above elbow, reactive hyperemia was induced by 5 min of distal lower arm occlusion by a blood pressure cuff inflated to 50 mm Hg above systolic pressure. Subsequently, the longitudinal image of the artery was recorded continuously until 2 min after the cuff deflation for the maximal brachial artery diameter determination. Pulsed Doppler velocity signals were acquired for 20 seconds before cuff inflation and for 10 seconds after cuff deflation for the hyperemic flow (% increase in blood flow) determination. We have already estimated the reproducibility and accuracy of the measurement by a preliminary test using 10 subjects. The same person, who performed the FMD measurement of this study, had measured FMD of each subject twice at one hour interval. The interclass correlation coefficient was 0.98 and systematic error across these two measurements was 0.1%. BODY.METHODS.SERUM MEASUREMENTS: Whole blood samples were collected from the left arm, just after the FMD and hemodynamic measurements. They were separated immediately by centrifugation (1,200 g, 10 min) and were stored at -80°C until use. Glucose, triglycerides, and total cholesterol were measured by enzymatic procedures (Mitsubishi Chemical Medience Corporation, Tokyo, Japan). High-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) were measured by enzymatic procedures (Kyowa Medex Co., Ltd., Tokyo, Japan). Insulin was measured by Chemiluminescent immunoassay (ABBOTT JAPAN Co., Ltd, Tokyo, Japan). High-sensitivity C-reactive protein (hs-CRP) was measured by nephelometry using N-latex CRP-2 (Siemens Healthcare Japan, Tokyo, Japan). Malondialdehyde-modified LDL (MDA-LDL) was measured by two step ELISA (SEKISUI MEDICAL Co., Ltd., Tokyo, Japan) which uses monoclonal antibody recognizing MDA residues (ML25) and monoclonal antibody against apoB (AB16) to make the assay specific to MDA-LDL as described previously [18]. BODY.METHODS.URINE MEASUREMENT: For the urine 8-isoprostane determinations, a single voided urine sample was collected from each subject just after the blood sampling and was stored at -80°C until use. To 1000 μl (Creatinine < 50 mg/dl) or 500 μl (Creatinine ≥ 50 mg/dl) urine sample, 3H-8epi PGF2α (20 dpm/μL) 50 μL and 4% acetic acid 3 ml were added. After being stirred for 30 min, the sample was applied on an OASIS HLB solid phase extraction column (Nihon Waters K.K., Tokyo, Japan) preconditioned with 2 ml each of Ethylacetate and Acetonitryl and 3 ml of 4% acetic acid). After being washed with 15% acetonitry in Ehylacetate: hexane: 1-butanol = 15: 85: 1, the column was eluted with 2 ml ethylacetate:hexane = 85: 1. The eluted sample was dried under nitrogen, dissolved in 1 ml EIA buffer, and stirred for 30 min before being subjected to EIA assay (Cayman Chemical, Michigan, United States) as described previously [19]. The urine creatinine for normalization of 8-isoprostane concentration was measured by enzymatic procedures (Mitsubishi Chemical Medience Corporation, Tokyo, Japan). BODY.METHODS.STATISTICAL ANALYSIS: A power analysis for two-way within subjects ANOVA was performed as described [20] and showed that a total sample size of 12 subjects was required to detect an interaction between meals and prandial status with an α error of 0.05 and a power of 0.80. Data are presented as means ± standard deviations. Baseline characteristics were compared between the curry-first and the control-first group by unpaired t-test. Statistical analyses about the effects of curry on FMD and other parameters were performed by using two-way within subjects ANOVA (control/curry and fasting/postprandial). If the interaction was significant, post hoc Student's paired t-test was performed. Statistical significance was assumed if a null hypothesis could be rejected at P = 0.05. All analyses were performed with SPSS (version 22; IBM Japan, Ltd., Tokyo, Japan). BODY.RESULTS.BASELINE CHARACTERISTICS: Of the 18 subjects enrolled in the study, 4 subjects were excluded from the analysis for the follwoing reasons. One subject canceled after the first participation. The ultrasound images of another subject were technically unsuitable for the analysis, Another subjects had a sleeping problem on the night before the test day, and the other subject showed very low postprandial glucose level (<50 mg/dL). Table 3 summarizes the baseline characteristics of the remaining 14 subjects. All baseline characteristics did not differ significantly between the control-first and the curry-first group. Table 3 Baseline characteristics Parameters Total (n = 14) Control-first (n = 7) Curry-first (n = 7) Age (years) 45 ± 9 42 ± 7 48 ± 10 Body mass index (kg/m 2 ) 23.7 ± 2.7 23.0 ± 1.9 24.4 ± 3.3 Systolic blood pressure (mm Hg) 112 ± 8 112 ± 5 112 ± 11 Diastolic blood pressure (mm Hg) 75 ± 7 76 ± 5 74 ± 9 Heart rate (beats/min) 60 ± 10 59 ± 7 61 ± 13 Total cholesterol (mg/dL) 196 ± 34 194 ± 34 199 ± 37 Triglycerides (mg/dL) 97 ± 44 99 ± 53 96 ± 38 HDL-cholesterol (mg/dL) 58 ± 15 58 ± 14 57 ± 17 LDL-cholesterol (mg/dL) 118 ± 28 114 ± 29 121 ± 28 Glucose (mg/dL) 90 ± 7 88 ± 6 91 ± 8 Insulin (μU/mL) 4.1 ± 1.9 3.9 ± 1.5 4.3 ± 2.4 hs-CRP (mg/dL) 0.122 ± 0.179 0.094 ± 0.176 0.149 ± 0.192 MDA-LDL (U/L) 88 ± 20 83 ± 20 93 ± 21 8-isoprostane (pg/mg Cr) 285 ± 81 276 ± 95 294 ± 70 Values are means ± SD. Abbreviations : HDL high-density lipoprotein, LDL low-density lipoprotein, hs-CRP high-sensitivity C-reactive protein, MDA malondialdehyde-modified, Cr creatinine. BODY.RESULTS.EFFECTS OF CURRY CONSUMPTION ON FMD AND OTHER HEMODYNAMIC PARAMETERS: Table 4 summarizes the brachial and hemodynamic parameters with significance probability values (P values) from the ANOVA results. Table 4 Vascular function and Systemic Hemodynamics   Control Curry   P values a   Parameters Fasting Postprandial Fasting Postprandial Meal effects Interaction Prandial effects FMD (%) 5.8 ± 2.4 5.1 ± 2.3 5.2 ± 2.5 6.6 ± 2.0 0.20 < 0.001 0.25 Systolic blood pressure (mm Hg) 110 ± 7 110 ± 10 111 ± 8 113 ± 7 0.07 0.57 0.33 Diastolic blood pressure (mm Hg) 73 ± 8 67 ± 7 75 ± 7 70 ± 7 0.07 0.32 < 0.001 Heart rate (beats/min) 60 ± 10 66 ± 11 59 ± 9 66 ± 10 0.56 0.55 < 0.001 Baseline arterial diameter (mm) 4.00 ± 0.47 4.12 ± 0.49 3.96 ± 0.47 4.02 ± 0.46 0.12 0.07 < 0.001 Peak hyperemic flow (%) 595 ± 259 536 ± 157 609 ± 217 571 ± 170 0.60 0.76 0.22 Values are means ± SD. Abbreviations : FMD flow-mediated vasodilation. a P values were calculated by using two-way within subjects ANOVA (control/curry and fasting/postprandial). Only on FMD, the interaction effect between meals (control/curry) and prandial status (fasting/postprandial) was significant (P < 0.001). Post hoc Student's paired t-tests revealed that the consumption of the control significantly decreased the FMD from 5.8 ± 2.4% to 5.1 ± 2.3% (Figure 1, P = 0.039). On the other hand, the consumption of the curry increased FMD from 5.2 ± 2.5% to 6.6 ± 2.0% (P = 0.001), and the postprandial FMD after the curry meal was higher than that after the control meal (5.1 ± 2.3% vs. 6.6 ± 2.0%, P = 0.002). Figure 1Effects of a single consumption of curry on flow-mediated vasodilation (FMD), n = 14. Because the interaction between meals (control/curry) and prandial status (fasting/postprandial) was found significant (P = 0.002) by using two-way within subjects ANOVA, P values were calculated by post hoc Student's paired t-test. Abbreviations: FMD flow-mediated vasodilation. After consumption of the test meals, heart rate and baseline arterial diameter increased significantly, and diastolic blood pressure decreased significantly. Other than these, no main effects of the meals, the prandial status, or no interaction effects on systemic and forearm hemodynamics were significant. BODY.RESULTS.EFFECTS OF CURRY CONSUMPTION ON BIOCHEMICAL PARAMETERS: Table 5 summarizes the biochemical parameters with P values from the ANOVA results. Table 5 Biochemical parameters   Control Curry   P values a   Parameters Fasting Postprandial Fasting Postprandial Meal effects Interaction Prandial effects Total cholesterol (mg/dL) 203 ± 36 195 ± 34 197 ± 32 191 ± 28 0.40 0.36 0.001 Triglycerides (mg/dL) 107 ± 59 114 ± 59 97 ± 44 103 ± 43 0.23 0.96 0.025 HDL-cholesterol (mg/dL) 58 ± 16 56 ± 16 59 ± 15 56 ± 15 0.55 0.53 < 0.001 LDL-cholesterol (mg/dL) 122 ± 30 118 ± 28 118 ± 27 112 ± 23 0.36 0.42 0.001 Glucose (mg/dL) 89 ± 7 113 ± 24 90 ± 7 117 ± 29 0.25 0.44 0.001 Insulin (μU/mL) 3.9 ± 2.0 26.0 ± 11.7 4.3 ± 1.9 25.5 ± 9.9 0.98 0.68 < 0.001 hs-CRP (mg/dL) 0.107 ± 0.159 0.104 ± 0.158 0.089 ± 0.145 0.087 ± 0.143 0.66 0.87 0.038 MDA-LDL (U/L) 94 ± 24 85 ± 24 95 ± 26 85 ± 21 0.93 0.91 0.027 8-isoprostane (pg/mg Cr) 273 ± 76 283 ± 89 277 ± 69 324 ± 67 0.23 0.17 0.047 Values are means ± SD. Abbreviations : HDL high-density lipoprotein, LDL low-density lipoprotein, hs-CRP high-sensitivity C-reactive protein, MDA malondialdehyde-modified, Cr creatinine. a P values were calculated by using two-way within subjects ANOVA (control/curry and fasting/postprandial). While the main effects of meals and the interaction effects between meals (control/curry) and prandial status (fasting/postprandial) were not significant, the main effects of prandial status were significant for all parameters measured. BODY.DISCUSSION: In the present study, we demonstrated that a single consumption of a dish of curry and rice improved the postprandial FMD in healthy men. We believe the significant interaction effect on FMD between meals and prandial status was due to the antioxidant components in the curry, although consumption of the curry meal did not change the two oxidative stress parameters (MDA-LDL and urine 8-isoprostane) measured in our study. Many researchers reported that FMD was improved by some antioxidant consumption (long term and short term) without 8-isoprostane decrease [21-23]. Few intervention studies using antioxidants report serum MDA-LDL levels. Pfeuffer et al. reported that consumption of 150 mg/d quercetin for 8 weeks affected neither oxidized LDL nor 8-isoprostane [24]. These reports suggest that no change in MDA-LDL and urine 8-isoprostane does not necessarily mean that improvement in FMD is not attributable to decrease in oxidative stress. On the other hand, it has been reported that administration of glucose in oral glucose tolerance test (OGTT) impaired endothelial function with concomitant increase in postprandial oxidative stress [11]. It is also reported that the postprandial serum glucose after rice is lower than that after the equivalent amount of glucose consumption [25]. In our study, we found that the test meals containing 82 g carbohydrate, which is nearly equivalent to the amount of glucose administered in 75-g OGTT, increased postprandial serum glucose and a consumption of the control meal resulted in decrease in FMD suggesting impairment of endothelial function. We speculate that the presence of spice antioxidants in the curry would have prevented the increase in the oxidative stress induced by postprandial serum glucose increase, but the change in the oxidative stress parameters after our test meal was too small to be detected, because the postprandial serum glucose level after our test meals (113 mg/dL after control or 117 mg/dL after curry) was much lower than that normally encountered after 75-g oral glucose loading [26]. Curcumin, eugenol and quercetin in curry could have contributed to our results suggesting that spices improved postprandial endothelial function. It was reported that curcumin alleviates an endothelium-dependent vasodilator dysfunction induced by high glucose in rat aortic rings and increased heme oxygenase-1 activity, and that stimulation of guanylyl cyclase may be involved in the protective effects of curcumin [27]. Eugenol was reported to produce smooth muscle relaxation resulting from the blockade of both voltage-sensitive and receptor-operated channels that are modulated by endothelial-generated nitric oxide [4]. It has been also suggested that qurcetin, which is a main polyphenol of onion and is also contained in curry, is known as a selective modulator of cyclic GMP-dependent relaxations [28]. Future studies are needed for elucidating the mechanism of our findings. After consumption of the test meals, baseline arterial diameter increased significantly. Similar increase in baseline arterial diameter after a fatty meal consumption has been reported [29,30]. Although many researchers reported that increase in baseline diameter would decrease FMD [31,32], the increase did not appear to have diminished the effect of spices on FMD, because significant interaction effect between meals and prandial status on baseline diameter was not found, and because the postprandial FMD after curry even increased despite that the increase of baseline diameter should decrease FMD. It is reported that a single consumption of a high antioxidant spice blend attenuated postprandial insulin and triglyceride responses and increased some plasma measures of antioxidant activities [33]. The results of the present study, however, showed that no interaction effects (i.e., the effects due to spices) on biochemical data between meals (control/curry) and prandial status (fasting/postprandial) were significant. In the previous study, the subjects were given a test meal with different composition, and with different kind of spices. Moreover, they collected blood samples at 30-min intervals for 3.5 h. These differences in experimental conditions may have affected the results. We found that postprandial diastolic blood pressure decreased and postprandial heart rate increased significantly, but postprandial systolic blood pressure did not change significantly. Berry et al. reported that glucose solution with ground beef (i.e., solid food), when fed to human subjects, caused higher postprandial systolic blood pressure than glucose solution alone (i.e., liquid food) [34]. They also reported that diastolic blood pressure fell and heart rates increased, without significant (observable) effects from the ground beef [34]. Although the mechanisms were not fully understood, the results of our study in which participants consumed solid meal, agree with their findings. There are some limitations in the current study. The first limitation is our small sample size (n = 14). Follow-up work using a larger sample size may be needed. The second limitation is that because of the unique flavor and color of spices used in the curry, the curry meals were readily distinguishable from the control meals by the subjects. Although we cannot exclude the possibility entirely that this feature of our study had some influence on our results, it is more likely that the presence of spices in the curry reduced the impact of the postprandial state on the endothelium after the meal. BODY.CONCLUSION: Curry consumption ameliorates postprandial endothelial dysfunction and may be beneficial for preventing cardiovascular events. Lifestyle-related diseases such as atherosclerosis and diabetes mellitus have become serious health problems in the modern world. Curry may be helpful in the fight against those lifestyle-related diseases. BODY.ABBREVIATIONS: FMD: Flow-mediated vasodilation; HDL-C: High-density lipoprotein-cholesterol; LDL-C: Low-density lipoprotein-cholesterol; hs-CRP: High-sensitivity C-reactive protein; MDA-LDL: Malondialdehyde-modified LDL; OGTT: Oral glucose tolerance test; Cr: Creatinine. BODY.COMPETING INTERESTS: This study was supported by House Foods Corporation. HN, SY and SS are employees of House Foods Corporation. NT, HS, and NM are employees of House Foods Group Inc. YH has no competing interest. BODY.AUTHORS’ CONTRIBUTION: HN designed the research, performed measurements and statistical analysis, and wrote the manuscript. NT conducted the research. HS provided statistical consultation and critically revised the manuscript. NM provided much consultation about the study design and conducted the research. SY designed and prepared test meals. SS provided much consultation about the study design. YH provided much consultation about the study design, conducted the research, provided consultation about the discussion of the test results, and critically revised the manuscript. All authors read and approved the final manuscript.

TITLE: A randomized, double-blind comparison of OROS ABSTRACT.BACKGROUND: Long-acting opioid formulations are advocated for maintaining pain control in chronic cancer pain. OROS® hydromorphone is a sustained-release formulation of hydromorphone that requires dosing once daily to maintain therapeutic concentrations. The objective of this study was to demonstrate the clinical equivalence of immediate-release and sustained-release formulations of hydromorphone and morphine for chronic cancer pain. ABSTRACT.METHODS: 200 patients with cancer pain (requiring ≤ 540 mg/d of oral morphine) participated in this double-blind, parallel-group trial. Patients were randomized to receive hydromorphone or morphine (immediate-release for 2–9 days, sustained-release for 10–15 days). Efficacy was assessed with the Brief Pain Inventory (BPI), investigator and patient global evaluations, Eastern Cooperative Oncology Group performance status, and the Mini-Mental State Examination. The primary endpoint was the 'worst pain in the past 24 hours' item of the BPI, in both the immediate-release and sustained-release study phases, with treatments deemed equivalent if the 95% confidence intervals (CI) of the between-group differences at endpoint were between -1.5 and 1.5. No equivalence limits were defined for secondary endpoints. ABSTRACT.RESULTS: Least-squares mean differences (95% CI) between groups were 0.2 (-0.4, 0.9) in the immediate-release phase and -0.8 (-1.6, -0.01) in the sustained-release phase (intent-to-treat population), indicating that the immediate-release formulations met the pre-specified equivalence criteria, but that the lower limit of the 95% CI (-1.6) was outside the boundary (-1.5) for the sustained-release formulations. BPI 'pain now PM' was significantly lower with OROS® hydromorphone compared with controlled-release morphine (least-squares mean difference [95% CI], -0.77 [-1.49, -0.05]; p = 0.0372). Scores for other secondary efficacy variables were similar between the two sustained-release treatments. At endpoint, > 70% of investigators and patients rated both treatments as good to excellent. The safety profiles of hydromorphone and morphine were similar and typical of opioid analgesics. ABSTRACT.CONCLUSION: Equivalence was demonstrated for immediate-release formulations of hydromorphone and morphine, but not for the sustained-release formulations of OROS® hydromorphone and controlled-release morphine. The direction of the mean difference between the treatments (-0.8) and the out-of-range lower limit of the 95% CI (-1.6) were in favor of OROS® hydromorphone. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov: NCT0041054 BODY.BACKGROUND: Opioid analgesics are highly effective for the treatment of pain, enabling 85% to 95% of patients to gain functional control of their lives [1,2]. Opioid therapy is typically initiated after the failure of maximum doses of non-opioid analgesics [3] and is titrated to attain the best balance between pain relief and side effects for each patient. The European Association for Palliative Care [4] and the American Pain Society [5] advocate the use of long-acting oral agents for maintaining analgesia once individual dose requirements have been established. For these reasons, long-acting opioids have become the mainstay of chronic cancer pain therapy. OROS® hydromorphone is a unique long-acting opioid formulation that utilizes Push-PullTM active osmotic technology developed by ALZA Corporation (Mountain View, CA, USA). The Push-PullTM system maintains consistent hydromorphone plasma concentrations throughout the 24-hour dosing interval, providing long-lasting analgesia [6-8]. Release of the drug from the system is actively controlled by the dosage form itself, and is not significantly influenced by environmental factors such as the surrounding pH or gastric motility [9,10]. There is a minimal effect of food on the rate and extent of absorption of hydromorphone from OROS® hydromorphone; in one study the mean geometric ratios of fed and fasted subjects for peak plasma concentration (Cmax) and area under the concentration-time curve (AUC) were within 20%; the median time to peak plasma concentration (Tmax) was lower under fed conditions (12 versus 16 hours), but mean plasma concentration profiles generally overlapped, especially up to 6 hours after dosing [11]. The pharmacokinetics of OROS® hydromorphone are also minimally affected by alcohol; one study found that plasma hydromorphone concentrations were slightly higher after alcohol (240 mL solutions of 4%, 20%, and 40% alcohol and orange juice) compared with no alcohol, but there was no clear alcohol dose-response relationship and no dose dumping of hydromorphone occurred [12]. The primary objective of the current study was to demonstrate the clinical equivalence of hydromorphone and morphine (immediate-release [IR] and sustained-release [SR] formulations) using the 'worst pain in the past 24 hours' item of the Brief Pain Inventory (BPI). Morphine was selected as the active comparator since it is the gold standard for pain control. Controlled-release (CR) morphine is available in twice-daily and once-daily formulations and is widely used to alleviate cancer pain; in this study the twice-daily formulation (dosing every 12 hours) was used. BODY.METHODS.PATIENTS: This study was a multicenter, phase III, randomized, double-blind (double-dummy), active-controlled, parallel-group, equivalence trial. It was conducted at 37 centers in Belgium, Canada, France, Germany, The Netherlands, Spain, Sweden, and the United Kingdom. The study enrolled inpatients, outpatients, and day-patients ≥ 18 years of age who had moderate to severe chronic cancer pain requiring 60 to 540 mg of oral morphine (or equivalent) every 24 hours. The criteria used for patient selection are listed in Table 1. Concomitant chemotherapy or radiotherapy was permitted. All patients who entered the trial were informed of the nature of the study, and provided written informed consent for participation. The study was conducted in accordance with the principles of the Declaration of Helsinki. Table 1 Criteria for patient selection Inclusion criteria Exclusion criteria • Age ≥ 18 years • Pain not considered potentially responsive to opioids • Presence of chronic cancer pain: • Pain present only upon movement  ◦ currently receiving strong oral or transdermal opioid analgesics (60–540 mg oral morphine or equivalent every 24 hours) • Need for other opioid analgesics (except study medication and breakthrough pain medication) after randomization  ◦ appropriate candidate for strong oral or transdermal opioid analgesics (anticipated requirement, 60–540 mg oral morphine or equivalent every 24 hours) • Current or recent (within 6 months) history of drug and/or alcohol abuse • Pain suitable for treatment with a once-daily formulation • Women of childbearing potential who are pregnant or lactating, seeking pregnancy, or failing to take adequate contraceptive precautions • Intolerance of, or hypersensitivity to, hydromorphone or other opioids • Presence of GI disease of sufficient severity to likely interfere with oral analgesia (e.g., dysphagia, vomiting, no bowel movement or bowel obstruction due to impaction within 5 days of study entry, severe gut narrowing that may affect analgesic absorption or transit) • Use of monoamine oxidase inhibitors within 2 weeks prior to study entry • Investigational drug use within 4 weeks of study entry • Presence of conditions for which risks of opioid use outweigh potential benefits (e.g., raised intracranial pressure, hypotension, hypothyroidism, asthma, reduced respiratory reserve, prostatic hypertrophy, hepatic impairment, renal impairment, elderly and debilitated, convulsive disorders, Addison's disease) Abbreviation: GI, gastrointestinal BODY.METHODS.DRUGS AND DOSAGES: Patients were randomized 1:1, with a central computer-generated randomization list, to receive hydromorphone or morphine for up to 24 days (Figure 1). This study consisted of 2 phases: an initial IR phase and a subsequent SR phase. In the IR phase, patients received IR formulations of either hydromorphone [Dilaudid®, Abbott Laboratories] or morphine (morphine sulfate IR [Sevredol®, Napp Laboratories]) every 4 hours (6 times daily) for 2 to 9 days. Patients underwent conversion from previous therapy to 1 of 6 possible initial dose levels, based on individual patient characteristics and generally accepted morphine equivalent conversion factors. Doses were selected according to the available tablet strengths and a working conversion ratio of 1:5 (1 hydromorphone:5 morphine equivalents), in the ranges hydromorphone IR 12–108 mg/day and morphine IR 60–540 mg/day. The dose was titrated to the next higher dose level if the patient had more than 3 breakthrough pain episodes requiring breakthrough pain medication within the previous 24 hours. Daily doses were titrated up to the next higher dose level, with no dose levels skipped, at most once a day. Dose titration was continued until dose-stable pain control was achieved. Patients who experienced 2 consecutive days with no more than 3 breakthrough pain episodes requiring rescue medication per day were considered to have achieved dose-stable pain control and could begin the SR phase of the study. Patients who did not achieve dose stable pain control by day 9 were withdrawn from the study, and end-of-study evaluations were carried out. Figure 1Study design. BID, twice daily; CR, controlled-release; IR, immediate-release; QD, once-daily; q4h, every 4 hours. The duration of the SR phase was 10 to 15 days. In this phase, patients continued to receive the same study drug but in an SR formulation: OROS® hydromorphone once-daily or CR morphine (morphine sulfate SR [MST Continus®, Napp Laboratories]) twice-daily. They were started on the same dose level number on which they had achieved dose-stable pain control in the preceding IR phase, which was then adjusted as required, at most every 2 days, in single steps so that no dose level was skipped. Doses were selected according to the available tablet strengths and a working conversion ratio of approximately 1:5 (hydromorphone:morphine), in the ranges OROS® hydromorphone 16–96 mg/day and CR morphine 60–520 mg/day. The SR phase was completed after a minimum of 10 days (maximum of 15 days) on OROS® hydromorphone or CR morphine if pain was controlled and the dose was stable for at least the previous 2 days. In the IR phase, the dosing regimen was the same for both drugs: patients received a dose of study medication 6 times a day, every 4 hours, at 0600, 1000, 1400, 1800, 2200, and 0200. In this phase, all treatments were over-encapsulated resulting in identical capsules. In the SR phase, dosing was done twice each day at 1000 and 2200. To maintain the blind in this phase, matching placebo tablets and capsules were used: patients in the OROS® hydromorphone group took OROS® hydromorphone and placebo CR morphine in the morning and placebo CR morphine in the evening; patients in the CR morphine group took CR morphine and placebo OROS® hydromorphone in the morning and CR morphine in the evening. In both study phases, patients could also receive breakthrough pain medication as needed, either hydromorphone or morphine, supplied as the IR formulation. A single dose of breakthrough pain medication contained approximately one-sixth of the patient's daily dose. BODY.METHODS.ASSESSMENTS: Baseline efficacy assessments were done before dosing on day 1 of the IR phase and included: the BPI (12-item instrument to assess pain intensity [from 0 = no pain to 10 = pain as bad as you can imagine], pain relief [0% to 100%], and pain interference with various aspects of the patient's life [from 0 = no interference to 10 = complete interference]) [13], the Mini-Mental State Examination (MMSE; 0–30, higher scores indicate better cognitive performance) [14], and the Eastern Cooperative Oncology Group (ECOG) performance status score (0–4, higher scores indicate poorer performance) [15]. Primary equivalence of efficacy was assessed using the BPI assessment of 'worst pain in the past 24 hours' on an 11-point scale ranging from 0 (no pain) to 10 (worst pain imaginable). This was selected as the primary efficacy measure because evidence suggests that severely intense pain is what interferes most significantly with activities of daily life [13]. Secondary endpoints included other assessments of pain from the BPI (completed by the investigator in consultation with the patient); investigator and patient global assessments of treatment effectiveness (1 = poor, 2 = fair, 3 = good, 4 = very good, 5 = excellent); MMSE; ECOG; time to dose stabilization; and usage and dose of breakthrough pain medication. Patients rated BPI 'worst pain in the past 24 hours' once daily (just before taking the mid-morning dose of study medication) and 'pain now' twice daily (AM and PM; just before taking the mid-morning and evening doses) throughout the study and recorded responses in daily diaries. The other BPI assessments, plus investigator and patient global evaluations of treatment effectiveness, MMSE, and ECOG, were evaluated at the end of both phases. Adverse events (AEs) and concomitant medications were recorded throughout the study. Physical examinations were carried out at baseline and at the end of each study phase. Blood and urine samples were collected for laboratory analysis (hematology, serum biochemistry, and urinalysis) at baseline and at the end of the SR phase. Patients were contacted daily during the IR phase and every 3 to 4 days during the SR phase (preferably by home or clinic visit, or by telephone if appropriate). Contact was also made 3 days after the last dose of study drug to determine the patient's AE status. BODY.METHODS.STATISTICAL ANALYSIS: Assuming variability (standard deviation, SD) in the primary efficacy variable of 2.0 [16] and a 30% dropout/non-evaluable rate, a sample size of approximately 70 patients per treatment group was required (140 total). This would provide 90% power to detect equivalence with an equivalence limit of 1.5. A planned blinded re-estimation of the optimal sample size was done by an appointed independent person after 55 patients had completed the study, the variability for the primary efficacy measure was estimated using their data, and the sample size was increased to 170 patients. Subsequently, an unplanned, blinded re-estimation of the variability of the primary variable for both phases was done after 120 patients had completed the study, and as a result, the sample size was increased to 200 patients. All statistical analyses were pre-specified. All efficacy variables were analyzed using the intent-to-treat (ITT) population, which included all patients who took at least 1 dose of study medication and had at least 1 assessment from each study phase. A per-protocol (PP) analysis, excluding data from patients with major protocol deviations, was also done. All patients taking at least 1 dose of study medication were included in the analysis of safety. All efficacy analyses included a pre-specified adjustment for baseline value and country to control for potentially confounding variables. The primary efficacy analysis used the mean of the last 2 post-baseline recorded values (or the last value if only 1 was available) for BPI 'worst pain in the past 24 hours'. The primary measure was calculated separately for the IR and SR phases (there were therefore two primary outcomes). An analysis of covariance (ANCOVA) was used to calculate the 95% two-sided confidence interval (CI) for the difference between the adjusted means for the 2 treatments at endpoint of each phase. An equivalence test was used to compare the mean scores of the primary efficacy measure at the end of each phase [17-19]. The 2 treatments, within a phase, were considered equivalent if the 95% two-sided CI for the treatment difference was within a -1.5 and 1.5 point difference on the BPI scale. This particular CI range was selected prospectively because it was considered clinically relevant through consultation with physicians who specialize in analgesia. For secondary endpoints (including BPI 'pain at its least in past 24 hours', 'pain on average pain', 'pain now' (AM and PM), 'pain relief in past 24 hours', 'pain interference in past 24 hours', MMSE, and ECOG) an ANCOVA model was used to calculate 95% two-sided CIs for the difference between the adjusted treatment means at the end of the IR and SR phases. The adjusted means were estimated by the same approach used for the primary endpoint, with the baseline score designated as a covariate; however, no equivalence limits were specified for the secondary endpoints. Time to dose stabilization during both study phases was analyzed by the log-rank test. Summary statistics with 95% CIs were produced for investigator and patient global evaluations of treatment effectiveness and to assess the number of medication doses taken for breakthrough pain in the last 2 days of each phase. P values (not adjusted for multiple comparisons) are reported for secondary endpoints. Statistical significance was declared at the α = 0.05 level. BODY.RESULTS.STUDY POPULATION: 202 patients were enrolled into the study (Belgium, n = 29; Canada, n = 10; France, n = 7; Germany, n = 15; Netherlands, n = 29; Spain, n = 41; Sweden, n = 11; UK, n = 60). 200 patients were randomized and took study medication. 163 patients (81.5%) completed the IR phase and 133 (66.5%) completed the SR phase. Figure 2 is a flow diagram of patient entry and completion and the reasons for premature discontinuation. Withdrawal from the study owing to lack of efficacy was more common in patients randomized to hydromorphone (n = 11) compared with morphine (n = 4). The mean age of participants overall was 59.8 years. 98.5% of patients were Caucasian and 51% were female. The most common types of cancer were breast (28%), lung (20%), and gastrointestinal (16%). For 133 (67%) patients, the predominant pain location was bone or soft tissue; 34 (17%) others had mixed pain, and 33 (17%) had visceral pain. No patient had neuropathic pain. The most common pain medications used at study entry were morphine (n = 124 [62%]) and tramadol (n = 38 [19%]). The baseline demographics and clinical characteristics were similar for both treatment groups (Table 2). Table 2 Demographic and baseline clinical characteristics Characteristic Hydromorphone (N = 99) Morphine (N = 101) Mean (SD) age, years 60.7 (12.50) 59.0 (11.36) Gender, % male 46.5 51.5 Race, % Caucasian/Black/Asian/Other 100/0/0/0 97/0/2/1 Mean (SD) height, cm 166.7 (9.33) 167.3 (10.47) Mean (SD) weight, kg 66.3 (15.33) 67.4 (13.33) Mean (SD) BMI, kg/m 2 23.8 (5.14) 24.2 (5.06) Cancer type, n (%)  Breast 23 (23.2) 33 (32.7)  Lung 20 (20.2) 19 (18.8)  Genitourinary 18 (18.2) 12 (11.9)  Gastrointestinal 17 (17.2) 15 (14.9)  Oral cavity 3 (3.0) 3 (3.0)  Lymphoma 3 (3.0) 0  Leukemia 1 (1.0) 2 (2.0)  Bone 1 (1.0) 1 (1.0)  Other 13 (13.1) 16 (15.8) Predominant pain type, n (%)  Bone or soft tissue 61 (61.6) 72 (71.3)  Mixed 19 (19.2) 15 (14.9)  Visceral 19 (19.2) 14 (13.9) Mean (SD) MMSE score* 28.5 (2.3) 28.8 (2.0) Mean (SD) ECOG score 1.6 (0.8) 1.6 (0.9) Abbreviations: BMI, body mass index; ECOG, Eastern Cooperative Oncology Group; MMSE, Mini-Mental State Examination; SD, standard deviation *Hydromorphone, n = 75; morphine, n = 73 Figure 2Patient disposition. IR, immediate-release; SR, sustained-release. BODY.RESULTS.EFFICACY.BPI 'WORST PAIN IN THE PAST 24 HOURS' (PRIMARY ENDPOINT): In each study phase, mean values for BPI 'worst pain in the past 24 hours' decreased with both hydromorphone and morphine treatments (Table 3). The least-squares (LS) mean (95% CI) differences between the hydromorphone and morphine groups were 0.2 points (-0.4, 0.9) in the IR phase and -0.8 points (-1.6, -0.01) in the SR phase (ITT population). In the IR phase, the 95% CI was within the -1.5 to 1.5 equivalency range (-0.4 to 0.9), indicating that IR hydromorphone and IR morphine were equivalent in this primary efficacy outcome measure. In the SR phase, the upper limit of the 95% CI was within the 1.5 boundary (-0.01), but the lower limit was less than -1.5 (-1.6), indicating that the SR formulations were not equivalent but that the negative direction of the mean difference was in favor of OROS® hydromorphone (Figure 3). Results of the analysis of the primary efficacy variable were similar when using data from the PP population. Table 3 BPI scores at baseline, end of IR phase, and end of SR phase (ITT population) Variable Baseline* Mean (SD) End of IR phase † LS mean (SE) End of SR phase ‡ LS mean (SE) Worst pain  Hydromorphone 6.3 (2.7) 5.0 (0.3) 3.5 (0.3)  Morphine 6.2 (2.5) 4.8 (0.3) 4.3 (0.3) Least pain  Hydromorphone 2.7 (2.5) 1.8 (0.2) 1.8 (0.2)  Morphine 2.9 (2.7) 2.2 (0.2) 1.8 (0.2) Average pain  Hydromorphone 5.0 (2.1) 3.6 (0.2) 3.4 (0.3)  Morphine 5.1 (2.1) 3.6 (0.2) 3.2 (0.3) Pain now AM  Hydromorphone 4.0 (2.7) 3.3 (0.3) 2.4 (0.3)  Morphine 4.1 (2.4) 3.4 (0.3) 2.8 (0.3) Pain now PM  Hydromorphone 4.8 (3.0) 3.6 (0.3) 2.6 (0.3) §  Morphine 4.2 (2.5) 3.7 (0.3) 3.4 (0.3) Abbreviations: BPI, Brief Pain Inventory; CR, controlled-release; IR, immediate-release; LS, least-squares; SD, standard deviation; SE, standard error; SR, sustained-release *Hydromorphone, n = 99; morphine, n = 101 †IR hydromorphone, n = 99; IR morphine, n = 101 ‡OROS ® hydromorphone, n = 77; CR morphine, n = 86 §p = 0.0372 versus CR morphine Figure 3Least-squares mean differences and 95% confidence intervals between the OROS® hydromorphone and controlled-release (CR) morphine groups at end of the sustained-release (SR) phase. BODY.RESULTS.EFFICACY.SECONDARY ENDPOINTS: At the end of the IR phase, mean scores for the secondary efficacy variables (other assessments of pain from the BPI [Table 3 and Figure 4], and MMSE and ECOG scores [data not shown]) were similar for IR hydromorphone and IR morphine, except for BPI 'pain interference with normal work', which favored IR hydromorphone (Figure 4; p = 0.0386). Figure 4Mean Brief Pain Inventory (BPI) interference scores at baseline, end of immediate-release (IR) phase, and end of sustained-release (SR) phase. Values at baseline represent means; values at other time points represent least-squares means. At baseline: hydromorphone, n = 99; morphine, n = 101. In IR phase: IR hydromorphone, n = 99; IR morphine, n = 101. In SR phase: OROS® hydromorphone, n = 77; controlled-release (CR) morphine, n = 86. At the end of the SR phase, scores were similar between treatment groups for BPI items 'least pain', 'average pain', and 'pain now AM'; however, 'pain now PM' was significantly lower in the OROS® hydromorphone group compared with the CR morphine group (LS mean [SE] scores, 2.6 [0.3] versus 3.4 [0.3], respectively; p = 0.0372) (Table 3). LS mean differences (95% CI) between treatment groups were 0.06 (-0.51, 0.64), 0.15 (-0.49, 0.79), -0.38 (-1.03, 0.27), and -0.77 (-1.49, -0.05) for least, average, morning, and evening pain, respectively (Figure 3). Figures 5 and 6 show the consistency of response to both OROS® hydromorphone and morphine throughout the SR phase, during both the morning and evening assessments of BPI 'pain now' scores. Figure 5Mean (standard error, SE) pain now scores in the morning during the sustained-release (SR) phase. Pain was rated using the 11-point Brief Pain Inventory (BPI), ranging from 0 (no pain) to 10 (worst pain imaginable). CR, controlled-release. Figure 6Mean (standard error, SE) pain now scores in the evening during the sustained-release (SR) phase. Pain was rated using the 11-point Brief Pain Inventory (BPI), ranging from 0 (no pain) to 10 (worst pain imaginable). CR, controlled-release. LS mean BPI 'pain relief' at endpoint was 69.7% for the OROS® hydromorphone group and 70.0% for the CR morphine group (95% CI for treatment difference, -8.11, 7.54). BPI 'pain interference' scores were also similar between treatment groups at endpoint, (Figure 4), as were LS mean ECOG performance status scores (1.8 versus 1.7 in the OROS® hydromorphone and CR morphine groups, respectively; 95% CI for treatment difference, -0.13, 0.26), and LS mean MMSE scores (28.9 versus 29.2; 95% CI for treatment difference, -1.19, 0.51). The majority of both investigators and patients (> 70%) rated the treatments as good, very good, or excellent at the end of each study phase (Figure 7). Mean ± SD patient global assessment scores at the end of the SR phase were 3.2 ± 1.14 for the OROS® hydromorphone group and 3.3 ± 0.98 for the CR morphine group (p = 0.6696); mean ± SD investigator global assessment scores were 3.2 ± 1.07 and 3.3 ± 0.91, respectively (p = 0.4760). Figure 7Patient (A) and investigator (B) global evaluations of treatment effectiveness at the end of the sustained-release (SR) phase. CR, controlled-release. In both treatment phases, patients in the hydromorphone group took longer than those in the morphine group to achieve stable doses (p < 0.001). The number of patients requiring dose-level changes during the SR phase was similar for both groups (19 of 77 [24.7%] OROS® hydromorphone recipients and 23 of 86 [26.7%] CR morphine recipients). Comparison of dosage levels at the end of the SR phase generally showed that similar proportions of patients received OROS® hydromorphone or CR morphine at each of the 5 (approximate 1:5 hydromorphone to morphine) lower dosage levels, although more patients in the OROS® hydromorphone group (10% versus 2% in the CR morphine group) received the highest dosage level evaluated in this study (Table 4). Table 4 Dose levels at end of SR phase OROS ® hydromorphone (N = 77) CR morphine (N = 86) Dose level Dose (mg) every 24 hours Number of subjects (%) Dose (mg) every 12 hours Number of subjects (%) 1 16 22 (28.6) 30 25 (29.1) 2 24 14 (18.2) 60 23 (26.7) 3 32 18 (23.4) 90 15 (17.4) 4 48 10 (13.0) 120 13 (15.1) 5 72 5 (6.5) 175 8 (9.3) 6 96 8 (10.4) 260 2 (2.3) Abbreviations: CR, controlled-release; SR, sustained-release In the IR phase, 61.6% of patients taking IR hydromorphone and 52.5% of those receiving IR morphine required medication for breakthrough pain on the last 2 days of the phase. In the SR phase, the corresponding percentages in the OROS® hydromorphone and CR morphine groups were 48.1% and 48.8%, respectively. The mean (SD) numbers of breakthrough pain medication doses taken on the last 2 days were as follows: IR phase, 1.8 (2.2) and 1.3 (1.8) for the IR hydromorphone and IR morphine groups, respectively; SR phase, 1.6 (2.2) and 1.4 (1.9) for the OROS® hydromorphone and CR morphine groups, respectively. The mean total doses of breakthrough pain medication as a percentage of the mean daily consumption were 39.4% and 27.1% for hydromorphone and morphine in the IR phase, and 13.8% and 10.3% for OROS® hydromorphone and CR morphine in the SR phase. There were no statistically significant treatment differences in the numbers of patients who took breakthrough pain medication or in the amount of medication taken in either phase. BODY.RESULTS.SAFETY: The overall safety profiles of hydromorphone and morphine were comparable, with a similar number of patients in each group reporting AEs. Overall, irrespective of study phase, 81 of 99 (81.8%) hydromorphone-treated patients and 90 of 101 (89.1%) morphine-treated patients reported at least 1 AE. The most commonly reported AEs during the IR and SR phases of the study are listed in Table 5. The types of AEs were typical of those generally associated with opioid use. The incidence of constipation was higher with hydromorphone than morphine in both phases of the study (23.2% versus 10.9% in the IR phase and 39.0% versus 22.1% in the SR phase). In the SR phase, the incidences of vomiting, nausea, and somnolence were higher with CR morphine than with OROS® hydromorphone (22.1% versus 9.1%, 29.1% versus 19.5%, and 14.0% versus 10.4%, respectively). Most AEs were mild or moderate in severity and no clear treatment difference was seen in the relative severity of the most commonly reported AEs. Approximately 30% of patients with AEs reported events that were considered unlikely to be related or not related to study therapy. Table 5 Adverse events reported by at least 5% of patients in any group during the IR or SR phase IR phase SR phase Adverse event Hydromorphone (N = 99) Morphine (N = 101) OROS ® hydromorphone (N = 77) CR morphine (N = 86) Constipation 23 (23.2%) 11 (10.9%) 30 (39.0%) 19 (22.1%) Nausea 18 (18.2%) 23 (22.8%) 15 (19.5%) 25 (29.1%) Vomiting 16 (16.2%) 19 (18.8%) 7 (9.1%) 19 (22.1%) Somnolence 11 (11.1%) 11 (10.9%) 8 (10.4%) 12 (14.0%) Dizziness 8 (8.1%) 6 (5.9%) 4 (5.2%) 8 (9.3%) Headache 8 (8.1%) 6 (5.9%) 3 (3.9%) 2 (2.3%) Diarrhea 8 (8.1%) 1 (1.0%) 7 (9.1%) 2 (2.3%) Pruritus 4 (4.0%) 5 (5.0%) 3 (3.9%) 5 (5.8%) Asthenia 5 (5.1%) 1 (1.0%) 6 (7.8%) 4 (4.7%) Fatigue 3 (3.0%) 3 (3.0%) 4 (5.2%) 6 (7.0%) Confusion 2 (2.0%) 2 (2.0%) 7 (9.1%) 2 (2.3%) Anemia 2 (2.0%) 1 (1.0%) 3 (3.9%) 6 (7.0%) Anorexia 1 (1.0%) 4 (4.0%) 2 (2.6%) 5 (5.8%) Insomnia 1 (1.0%) 2 (2.0%) 5 (6.5%) 4 (4.7%) Peripheral edema 0 3 (3.0%) 1 (1.3%) 8 (9.3%) Pyrexia 2 (2.0%) 1 (1.0%) 4 (5.2%) 2 (2.3%) Anxiety 1 (1.0%) 1 (1.0%) 5 (6.5%) 1 (1.2%) Abbreviations: CR, controlled-release; IR, immediate-release; SR, sustained-release Serious AEs were reported for 24 patients during the double-blind period (by 5 patients in each treatment group in the IR phase, and by 7 patients in the OROS® hydromorphone group and 9 in the CR morphine group in the SR phase [2 patients had serious AEs in both phases]). Many of the serious AEs were associated with the underlying disease, although approximately one third were considered definitely or probably related to study therapy. Three patients in the morphine group died during the study (2 during the IR phase, 1 during the SR phase). None of the deaths was considered related to the study medication. Twenty-six patients withdrew prematurely from the study because of AEs, 16 during the IR phase (9 hydromorphone, 7 morphine) and 10 during the SR phase (6 OROS® hydromorphone, 4 CR morphine). There was no statistically significant difference between treatment groups in the time to withdrawal, irrespective of phase (IR phase, p = 0.6537; SR phase, p = 0.2827). The use of concomitant medications, including the use of non-opioid analgesics, was similar for both treatment groups in both the IR and SR phases. BODY.DISCUSSION: This was a short-term, randomized, double-blind comparative study testing the clinical equivalence of IR and SR formulations of hydromorphone and morphine in patients with chronic cancer pain. In each study phase (IR and SR), mean values for BPI 'worst pain in the past 24 hours' (the primary efficacy endpoint) decreased with both hydromorphone and morphine. In the IR phase, the LS mean difference between the treatment groups was 0.2 points, and the 95% CI (-0.4, 0.9) was within the pre-specified equivalency range of -1.5 to 1.5. In the SR phase, the LS mean difference was -0.8 points in favor of OROS® hydromorphone. The upper limit of the 95% CI (-0.01) was within the 1.5 boundary, but the lower limit (-1.6) was less than the -1.5 limit. Therefore, according to the pre-specified criteria, equivalence was not demonstrated; the direction of the difference was in favor of OROS® hydromorphone. The BPI item 'worst pain in the past 24 hours' was selected as the primary endpoint of the trial based on previous studies showing it to be the most sensitive BPI measure in the clinical trial setting [13,20-22]. The results of the present study further suggest that OROS® hydromorphone provides consistent pain relief over 24 hours, and that pain levels in the evening were significantly lower after OROS® hydromorphone compared with CR morphine treatment. This finding reflects the 'pain now PM' measure being scored at the end of morphine dosing (i.e. trough levels) but at the midpoint of hydromorphone dosing. The comparable results between groups for the other secondary efficacy variables (including assessments of pain interference with physical activity and social function, performance status, and cognition) support similarity in the treatment efficacy. Withdrawal from the study owing to lack of efficacy was more common in patients randomized to hydromorphone (n = 11) compared with morphine (n = 4). This is a potentially negative finding for hydromorphone; however, the numbers are too low to draw any definite conclusions. Results of the dose analysis were generally consistent with those of previous reports, which indicate that most cancer pain can be controlled with ≤ 240 mg of oral morphine daily [23]. In the present study, 83% of the OROS® hydromorphone group and 88% of the CR morphine group received the equivalent of morphine 240 mg/day or less. The frequency of breakthrough medication use remained stable throughout both phases in the morphine group (52.5% in the IR phase, 48.8% in the SR phase), but decreased from the IR phase to the SR phase in the OROS® hydromorphone group (from 61.6% to 48.1%). At the end of the SR phase, there was no significant difference between the groups in use of breakthrough medication. The occurrence of 3 deaths and 23 serious AEs during the study was not unexpected, given the severity of the patients' conditions and the progressive nature of the disease. In fact, many of the serious AEs were associated with the underlying disease. The participants in this study are representative of the overall population afflicted with chronic cancer pain. The mean baseline BPI score for 'worst pain in the past 24 hours' was 6.3 on an 11-point scale, a score indicative of moderate to severe pain [16]. This is comparable to baseline values in other studies of cancer pain, for which the mean score was 6.99 [24]. Baseline cognition was similar to that of healthy controls (mean MMSE score, 28.3) [25], and the lack of change observed in both treatment groups probably reflects the fact that the MMSE was designed to measure cognition in Alzheimer's disease [14] and was not intended to measure cognition in non-demented subjects. The major positive aspect of this study was the robust design: it was multicenter, randomized, double-blind, and active controlled. It also used a widely utilized and well validated primary outcome measure, the BPI. On the negative side, there was a relatively poor completion rate, with only 60 out of 99 patients (60.6%) randomized to hydromorphone and 73 out of 101 patients (72.3%) randomized to morphine completing both treatment phases. However, this completion rate was reasonably good when taking into consideration that this study was carried out in patients with cancer which was advanced enough to require strong opioid analgesics. In addition, a dropout rate of at least 30% was anticipated. Furthermore, the duration of the study was relatively short (up to 24 days), so it was not possible to make evaluations of the sustained efficacy of the formulations. Two previous randomized, double-blind, crossover studies have compared CR formulations of hydromorphone and morphine in patients with cancer pain [26,27]. Moriarty et al (N = 100) found no significant differences between treatment groups for any outcome measure, and AEs were mild and infrequent [27]. In the other study (N = 87), patients treated with hydromorphone had significantly higher pain scores and required more doses of rescue analgesia than patients receiving morphine [26]. In addition, more patients in the hydromorphone group withdrew owing to AEs or inadequate analgesia (n = 16 versus n = 2 on morphine). Although it is difficult to directly compare the results of these studies with those of the current study, the observed differences may reflect, at least in part, the more consistent delivery of hydromorphone with the OROS® formulation relative to the dosing used in those studies. The consistent release of hydromorphone over 24 hours has been demonstrated in previous studies, in which steady-state plasma concentrations were achieved by 48 hours and sustained throughout the dosing interval [8,28]. Practicing clinicians are acutely aware of the need for multiple pain management alternatives, because it is clear that the response to a particular opioid agent varies among individuals. Although our understanding of the causes of individual differences remains incomplete, receptor and receptor-binding studies suggest that they may result not only from differences in the class of opioid receptors to which each agent binds (morphine binds to mu [primary] and kappa [lesser extent] [29]; hydromorphone binds to mu [primary] and delta [lesser extent] [30]), but also from inter-individual differences in opioid receptors (several receptor subtypes have been identified [mu 1, mu 2, mu 3, delta 1, delta 2, kappa 1, kappa 2, kappa 3]) and the location of these variants within the nervous system [31-33]. Therefore, clinicians may need to utilize different opioids to achieve maximum efficacy for each patient. In addition, some studies have supported the use of opioid rotation, switching from one opioid to another when treatment-limiting toxicity results in reduced responsiveness [34-37]. Based on numerous observations that individual response varies greatly from opioid to opioid, changing to a different drug may provide an improved balance between analgesia and adverse effects; as a result, there is a need for additional long-acting opioids on the market. Long-acting opioid formulations such OROS® hydromorphone have the potential to improve the overall management of chronic pain. Such formulations provide more consistent opioid plasma concentrations, avoiding the peaks and troughs associated with short-acting agents, and result in well-tolerated, around-the-clock pain control with fewer daily doses [38,39]. BODY.CONCLUSION: This study demonstrates that while IR hydromorphone is equivalent to IR morphine for relieving chronic cancer pain, equivalence was not demonstrated for OROS® hydromorphone and CR morphine in the SR phase of the study. The direction of the mean difference between the treatments, and the out-of-range lower limit of the 95% CI was in favor of OROS® hydromorphone on the primary endpoint 'worst pain'. 'Pain now PM' scores were also significantly lower for those taking OROS® hydromorphone at the end of the SR phase (although the timing of this measure coincided with a trough in the dosing levels for the twice-daily morphine compared with the once-daily OROS® hydromorphone), and BPI 'interference with normal work' scores were significantly lower for IR hydromorphone. None of the other secondary efficacy analyses showed significant treatment differences in either phase. The overall safety profiles of the 2 treatments were generally similar and most AEs were typical of opioid analgesic therapy. BODY.COMPETING INTERESTS: MH was a paid advisor for Janssen-Cilag. BODY.AUTHORS' CONTRIBUTIONS: MH was an investigator in this study and was involved in revising this manuscript for important intellectual content. JT contributed to the analysis of the study and reviewed the manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here:

TITLE: The implementation of a community-based aerobic walking program for mild to moderate knee osteoarthritis (OA): a knowledge translation (KT) randomized controlled trial (RCT): Part I: The Uptake of the Ottawa Panel clinical practice guidelines (CPGs) ABSTRACT.BACKGROUND: The implementation of evidence based clinical practice guidelines on self-management interventions to patients with chronic diseases is a complex process. A multifaceted strategy may offer an effective knowledge translation (KT) intervention to promote knowledge uptake and improve adherence in an effective walking program based on the Ottawa Panel Evidence Based Clinical Practice Guidelines among individuals with moderate osteoarthritis (OA). ABSTRACT.METHODS: A single-blind, randomized control trial was conducted. Patients with mild to moderate (OA) of the knee (n=222) were randomized to one of three KT groups: 1) Walking and Behavioural intervention (WB) (18 males, 57 females) which included the supervised community-based aerobic walking program combined with a behavioural intervention and an educational pamphlet on the benefits of walking for OA; 2) Walking intervention (W) (24 males, 57 females) wherein participants only received the supervised community-based aerobic walking program intervention and the educational pamphlet; 3) Self-directed control (C) (32 males, 52 females) wherein participants only received the educational pamphlet. One-way analyses of variance were used to test for differences in quality of life, adherence, confidence, and clinical outcomes among the study groups at each 3 month assessment during the 12-month intervention period and 6-month follow-up period. ABSTRACT.RESULTS: Short-term program adherence was greater in WB compared to C (p<0.012) after 3 months. No statistical significance (p> 0.05) was observed for long-term adherence (6 to 12 months), and total adherence between the three groups. The three knowledge translation strategies demonstrated equivalent long-term results for the implementation of a walking program for older individuals with moderate OA. Lower dropout rates as well as higher retention rates were observed for WB at 12 and 18 months. ABSTRACT.CONCLUSION: The additional knowledge translation behavioural component facilitated the implementation of clinical practice guidelines on walking over a short-term period. More studies are needed to improve the long-term walking adherence or longer guidelines uptake on walking among participants with OA. Particular attention should be taken into account related to patient's characteristic and preference. OA can be managed through the implementation of a walking program based on clinical practice guidelines in existing community-based walking clubs as well as at home with the minimal support of an exercise therapist or a trained volunteer. ABSTRACT.TRIAL REGISTRATION: Current Controlled Trials IRSCTNO9193542 BODY.BACKGROUND: Rising healthcare costs, limited resources, and the aging population have created new and growing challenges in the management of osteoarthritis (OA). The challenges associated with the management of OA are numerous as the prevalence of the population continues to increase while healthcare resources remain limited [1-7]. It is therefore necessary to determine the most effective methods of integrating research evidence in order to optimize health outcomes. The purpose of this randomized controlled trial (RCT) was to compare 1) improvements in quality of life (QoL) and clinical outcomes such as pain, mobility and endurance); 2) adherence rates; and 3) confidence and self-efficacy after the implementation of a 12-month supervised community-based aerobic walking program (SCAWP) based on the Ottawa Panel clinical practice guidelines (CPG) among three knowledge translation (KT) intervention arms. QoL, confidence, and self-efficacy were compared at 12- months (end of treatment) and at 18-months (6-months post-intervention). Adherence was compared during the intervention period at 3, 6, 9 and 12 months. The first part of this manuscript introduces each KT intervention and demonstrates the impact of knowledge (CPG) uptake of each by comparing outcomes influenced by the KT intervention such as adherene and behaviour change (confidence and self-efficacy). Knowledge application of each KT intervention is explained through the use a theoretical framework: Knowledge-To-Action Cycle (KTAC) [8,9]. The second part of this manuscript focuses on outcome evaluation, a specific phase in the KTAC framework used to guide this study. Part II demonstrates the effect of each intervention involving a walking program on outcomes which were influenced by the SCAWP including QoL (primary outcome) and other clinical outcomes such as pain, mobility, and endurance. These outcomes are exhibited in part II. Therefore, both parts of this manuscript were split according to the ''Evaluate outcomes" phase of the KTAC framework. Part I focused on KT outcomes which measured the success of CPG uptake through participants' adherence and behavioural change while part II focused on clinical outcomes which measured the positive effect of SCAWP due to the indirect CPG uptake/implementation/adoption by individuals with OA of the knee. BODY.BACKGROUND.KNOWLEDGE TRANSLATION: A major issue in health research today is finding effective and efficient ways to exchange knowledge between researchers, clinicians and the general public. Potential benefits to health which could be accrued through the application of research findings are not realized due to challenges in research uptake and knowledge translation [10]. KT is defined by Estabrooks et al. [11] as "the process by which specific research-based knowledge (science) is implemented in practice." The available evidence suggests that with an effective KT strategy, uptake of evidence-based clinical practice guidelines (EBCPGs) can be effective in improving patient health outcomes [12]. BODY.BACKGROUND.PHYSICAL ACTIVITY AND OSTEOARTHRITIS: The promotion of physical activity (PA) is a priority for health organizations serving the general population [13,14] and is highly recommended for subgroups affected by chronic diseases [15-19]. Community-based PA combined with a behavioural modification and self-management interventions can reduce the risk of disability and negative consequences of inactivity related to OA [20,21]. The challenge is to develop PA programs that will encourage OA patients to initiate and maintain improvements in exercise behaviour over a long-term period [22]. The available evidence suggests that with an effective KT strategy, uptake of evidence-based clinical practice guidelines (CPGs) can be effective in improving patient health outcomes [12]. BODY.BACKGROUND.SELECTING AN EFFECTIVE KT STRATEGY: Multifaceted interventions combining more than one KT strategy have tended to be more effective than using a single KT strategy alone [23,24] and have shown to have the greatest impact on CPG adherence and PA performance [25]. Behavioural interventions have been used for other chronic conditions to improve long-term adherence and maintenance of PA regimens with varying success. The efficacy of different behavioural interventions including patient education, health counselling, goal setting, and social/peer support, delivered separately and or in combination, have been examined in the management of arthritis [26-31]. BODY.METHODS: The following methodology is in full agreement with the Consolidated Standards of Reporting Trials (CONSORT) 2010 Statement criteria for reporting RCTs [32]. BODY.METHODS.DESIGN: This single blind RCT, funded by the Canadian institutes of Health Research, used a parallel group design (1:1:1). This community-based study was approved by the University of Ottawa Research Ethics Board and the City of Ottawa Public Health Research Ethics Board. This study implemented one of the Ottawa Panel CPG recommendations related to a supervised community-based aerobic walking programs (SCAWPs) among individuals with mild to moderate OA of the knee [33-35]. In order to facilitate an understanding of the KT process, Graham et al. developed a conceptual framework entitled "the Knowledge-To-Action Cycle" (Figure 1) which provides an approach which combines commonalities from various planned-action theories [8,9]. This framework was used to guide the development of the three KT strategies used in this RCT (Additional file 1). The framework demonstrates the dynamic process of knowledge creation and application [9]. Knowledge creation consists of three phases: knowledge inquiry, knowledge synthesis, and knowledge tools/products. The knowledge creation cycle of the framework demonstrates how knowledge is refined through each phase to provide useful information for end users. The action cycle of the framework consists of seven phases which may occur sequentially or simultaneously: 1) Identify the Knowledge-To-Action Cycle gaps; 2) Adapt Knowledge to local context; 3) Assess barriers to Knowledge Use; 4) Select, Tailor, Implement Interventions; 5) Monitor Knowledge Use; 6) Evaluate outcomes; and 7) Sustained Knowledge Use. For part I, the emphasis was placed on the "Evaluate outcomes (KT outcomes)" phase as participants' knowledge uptake was measured as adherence to the effective walking program for OA and attitude/behaviour change (self-efficacy and confidence) after participating in the SCAWP. Figure 1Knowledge-To-Action Cycle. This figure illustrates the Knowledge-To-Action Cycle Framework [8]. This figure is used with permission: Lost in knowledge translation: Time for a map. Graham ID, et al. Journal of Continuing Education in the Health Professions, 26(1). Copyright © 2006. The Alliance for Continuing Medical Education, The Society for Academic Continuing Medical Education, and The Association for Hospital Medical Education. The hypothesis was that the additional behavioural approach of the multifaceted KT intervention would: 1) increase short and long term adherence to the SCAWP; 2) reduce the drop-out rate; 3) influence behaviour change by improving self-efficacy, confidence, QoL and other clinical outcomes. BODY.METHODS.SAMPLE AND RECRUITMENT: A total of 222 adults with knee OA were recruited. Participants were eligible to participate in the study if he/she 1) had a confirmed diagnosis with mild to moderate unilateral or bilateral OA according to the American College of Rheumatology clinical and radiographic/magnetic resonance imagery criteria; 2) reported pain for at least 3 months; 3) expected his/her medication to change during the study period; 4) demonstrated an ability to ambulate for a minimum of 20 minutes, at their own pace with minimal reports of pain (≤ 3 out of 10 on a visual analogue pain rating scale) [36]; 4) were able to be treated as an out-patient; and 5) were available three times a week over a period of 12 months. Potential study subjects were excluded if they had: 1) Participated in regular physical or aerobic sports ≥ 2 times per week for more than 20 minutes per session during the previous 6 months; 2) severe OA of the knee or other weight bearing joints of the lower extremity; 3) no written consent from their physician to participate in the study; 4) pain at rest or at night; 5) received rehabilitation treatment, corticosteroids injection, or any other pain-related treatment besides medication for arthritis within the last 12 months; 6) uncontrolled hypertension (Systolic blood pressure >160 mm Hg confirmed by the screening initial VO2 max test at the Ottawa Heart Institute) 7) other illnesses, such as rheumatoid arthritis (judged by the patient or study physician to make participation in this study inadvisable); 8) significant cognitive deficit resulting in an inability to understand or comply with instructions; 9) surgery planned in the next year; 10) Intention to move away from Ottawa region in the next year; 11) an inability to communicate in English or French; or 12) an unwillingness to sign informed consent. The study coordinator assessed the inclusion/exclusion criteria of all potential participants by telephone. At the first visit, written informed consent was obtained if the patient met all inclusion criteria. BODY.METHODS.INTERVENTION: Participants were randomly assigned to one of the three KT intervention groups using central randomization [37] and computer generated numbers [38]: 1) Walking and Behavioural intervention (WB) (18 males, 57 females) which included the SCAWP with a behavioural intervention and an educational pamphlet on the benefits of walking for OA; 2) Walking intervention (W) (24 males, 57 females) wherein participants only received the SCAWP intervention and the educational pamphlet; 3) Self-directed control (C) (32 males, 52 females) wherein participants only received the educational pamphlet. All 3 groups were provided with pedometers and log books to be completed to measure physical performance (walking in minutes) and additional PA aside from the walking sessions (Table 1). The KT strategies were implemented over a 12-month duration and participants were assessed for additional 6 months (15 and 18-month follow-up assessments). Table 1 Summary of the KT strategies used KT strategies in each group WB or WalkingClub+ (supervised walking program + behavioural approach at the community-based Walking Club) W or WalkingClub (supervised walking program only at the community-based Walking Club) C or Self-directed Home-based or community based different than W and WB (unsupervised/self-directed Walking program only) EBP walking program (Ottawa Panel, 2005/2012) refs: [ 33 - 35 ] EBP walking program (Ottawa Panel, 2005/2012) refs: [ 33 - 35 ] Walking program (general info from pamphlet on walking and OA) Walking Club + effect (supervised program, team, daily monitored vital measures: BP, HR, #steps,) Walking Club effect (supervised program, team, daily monitored vital measures: BP, HR, #steps,) N/A Pedometer as measurement tool, but becomes a KT strategy Pedometer as measurement tool, but becomes a KT strategy Pedometer as measurement tool, but becomes a KT strategy Log book including 7-day PAR as measurement tool, but becomes a KT strategy Log book including 7-day PAR as measurement tool, but becomes a KT strategy Log book including 7-day PAR as measurement tool, but becomes a KT strategy $ compensation each walking session and at evaluation session $ compensation each walking session and at evaluation session $ compensation each logbook fulfilled and at evaluation session Study affiliation/participation (feel committed) Study affiliation/participation (feel committed) Study affiliation/participation (feel committed) Behavioural intervention (Baycrest PACE-ex: patient education + goal settings; PA counselling, telephone support) N/A N/A Following the successful randomization process, wherein no statistical difference was found between the three groups (with the exception of the "physical role" category of the SF-36: part II) (Tables 2, 3, 4, 5), research staff and evaluators were blinded to the treatment allocation. An independent evaluator was blinded to assess outcome measures. Due to the nature of the physical intervention, it was not practical to blind the study participants and PA specialists supervising the aerobic walking program [39]. Table 2 Subject demographics and baseline characteristics Characteristic Walking (n=79) Walking and Behavioural (n=69) Self-directed Control (n=74) Total Mean age (SD), yrs 63.9 (10.3) 63.9 (8.2) 62.3(6.8) 63.4 (8.6) Missing Data 0 0 0 0 Men/women, (%) 24 (30.4)/55 (69.9) 18 (26.1)/51 (73.9) 27 (36.5)/47 (63.5) 69 (31.1)/153 (68.9) Missing Data 0 0 0 0 Affected knee, n (%)         Right 33 (41.8) 31 (44.9) 25 (33.8) 89 (40.1) Left 31(39.2) 23 (33.3) 28 (37.8) 82 (36.9) Both side 15 (19.0) 15 (28.4) 21 (28.4) 51 (23.0) Missing Data 0 0 0 0 Mean duration of OA (SD), yrs 9.54 (8.09) 11.3 (9.7) 10.0 (9.9) 10.3 (9.26) Missing Data 0 0 0 0 Mean weight (SD), kg 80.7 (18.5) 83.1 (15.4) 83.0(15.8) 82.2 (16.6) Missing Data 0 1 0 0 Mean BMI (SD), kg/m² 29.4 (5.4) 30.3 (5.6) 29.9(5.3) 29.8 (5.4) Missing Data 0 2 4 6 Walking aid, n (%)         Yes 10 (12.7) 10 (14.5) 9 (12.2) 29 (13.1) No 69 (87.3) 58 (84.1) 64 (86.5) 191 (86.0) Missing Data 0 1 1 2 Racial background, n (%)         White 69 (87.3) 60 (87.0) 68 (91.9) 197 (88.7) Black 1 (1.3) 3 (4.3) 1 (1.4) 5 (2.3) Hispanic 2 (2.5) 2 (2.9) 4 (5.4) 8 (3.6) Asian or Pacific Islander 5 (6.3) 4 (5.8) 1 (1.4) 10 (4.5) American Indian or Alaskan native 1 (1.3) 0 (0) 0 (0) 1 (0.5) Other 1 (1.3) 0 (0) 0 (0) 1 (0.5) Missing Data 0 0 0 0 Marital status, n (%)         Married 46 (58.2) 36 (52.2) 44 (59.5) 126 (56.8) Separated 2 (2.5) 1 (1.4) 1 (1.4) 4 (1.8) Divorced 9 (11.4) 17 (24.6) 8 (10.8) 34 (15.3) Widowed 17 (21.5) 11 (15.9) 9 (12.2) 37 (16.7) Never Married 5 (6.3) 4 (5.8) 12 (16.2) 21 (9.5) Missing Data 0 0 0 0 Level of education, n (%)         Less than 7 yrs of school 2 (2.5) 1 (1.4) 1 (1.4) 4 (1.8) Grades 7 through 9 5 (6.3) 0 (0) 0 (0) 5 (2.3) Grades 10 through 11 7 (8.9) 4 (5.8) 5 (6.8) 16 (7.2) High school graduate 13 (16.5) 16 (23.2) 8 (10.8) 37 (16.7) 1 to 4 yrs of college 13 (16.5) 9 (13.0) 22 (29.7) 44 (19.8) College graduate 25 (31.6) 21 (30.4) 19 (25.7) 65 (29.3) Professional or Graduate school 14 (17.7) 18 (26.1) 19 (25.7) 51 (23.0) Missing Data 0 0 0 0 Yrs: years, OA: osteoarthritis, kg: kilograms, m: meters, %: validity percent. Table 3 Subject’s Medication at Baseline Medication Walking (W) only (n=79) Walking and Behavioural (WB) (n=69) Self-directed (C) (n=74) Oral hypoglycaemic agents         Total 2 (3) 5 (7) 9 (12)   Biguanide Class 2 (3) 4 (6) 9 (12)   Sulfonylureas Class 0 (0) 0 (0) 2 (3)   Thiazolidinedione 0 (0) 2 (3) 2 (3) Antihypertensive Agents         Total 22 (28) 15 (22) 15 (22)   Calcium channel blockers 6(8) 0(0) 7(9)   Antiotensin-converting enzyme 3 (4) 7 (10) 7 (9)   Angiotensin II receptor blockers 6 (8) 6 (9) 3 (4)   Beta-blocker 4 (5) 3 (4) 4 (5)   Alpha-blocker 3 (4) 0 (0) 0 (0)   Diuretic 5 (6) 8 (12) 5 (7) Antiarrhythmic 1 (1) 0 (0) 0 (0) Anti-anginal 1 (1) 0 (0) 0 (0) Anti-platelet agent 1 (1) 1 (1) 1 (1) Anticoagulant 0 (0) 2 (3) 0 (0) Phosphodiesterase type 5 inhibitors 0 (0) 1 (1) 0 (0) Statins 8 (10) 12 (17) 10 (14) Hormone         Total 11 (14) 10 (14) 11 (15)   Thyroid 5 (6) 5 (7) 8 (11)   Insulin 0 (0) 3 (4) 1 (1)   Corticosteroid 4 (5) 2 (3) 3 (4)   Progesterone 1 (1) 0 (0) 1 (1)   Androgen 1 (1) 0 (0) 0 (0)   Oestrogen 1 (1) 0 (0) 0 (0) Antithyroid 0 (0) 1 (1) 0(0) SERMs (Selective estrogen receptor modifiers) 1 (1) 0 (0) 0 (0) Beta 2-adrenergic receptor agonist (bronchodilator) 2(3) 1 (1) 3 (4) NSAIDs (Non-steroid anti-inflammatory drugs) 26 (33) 21 (30) 18 (24) COX-2 selective inhibitor 3 (4) 4 (6) 1 (1) NSAIDS       Betahistine 0 (0) 1 (1) 0 (0) DMARDs (Disease modifying antirheumatic drugs) 0 (0) 1 (1) 0 (0) Histamine antagonist 0 (0) 2 (3) 0 (0) Bisphosphonate 6 (8) 4 (6) 4 (5) Antibiotics 2 (3) 1 (1) 4 (5) Analgesics 1 (1) 1 (1) 3 (4) Opiates 0 (0) 0 (0) 2 (3) Hypnotic 4 (5) 3 (4) 4 (5) Antidepressant 7 (9) 5 (7) 7 (9) Psychostimulant 0 (0) 1 (1) 0 (0) Antipsychotic 0 (0) 1 (1) 0 (0) Anti-manic 0 (0) 1 (1) 0 (0) Anti-convulsant 1 (1) 1 (1) 0 (0) Protein pump inhibitor (PPI) 9 (11) 5 (6) 6 (8) Enzyme inhibitor 0 (0) 1 (1) 2 (3) Muscle relaxant 0 (0) 1 (1) 1 (1) Antispasmodic 0 (0) 1 (1) 0 (0) Anti-cholinergic 1 (1) 1 (1) 0 (0) Antineoplastic 2 (3) 1 (1) 0 (0) Antimetabolite 1 (1) 1 (1) 1 (1) Antifolate 0 (0) 1 (1) 1 (1) Latanoprost 0 (0) 1 (1) 0 (0) Viscosupplementation 0 (0) 1 (1) 0 (0) Xanthine 1 (1) 0 (0) 0 (0) Antifungals 0 (0) 0 (0) 1 (1) Antimalarial 0 (0) 1 (1) 1 (1) Nitrate 0 (0) 0 (0) 2 (3) Laxative 0 (0) 0 (0) 2 (3) Supplements 22 (28) 22 (28) 18 (24) Homeopathic Medication 1 (1) 1 (1) 1 (1) None 11 (14) 10 (13) 9 (12) Not Specific 21 (27) 21 (27) 20 (27) Missing Data         Total 4 (5) 8 (10) 17 (23)   No Evaluation* 2 (3) 4 (5) 7 (9)   No File** 2 (3) 4 (6) 10 (14) This table presents a list of medications which participants declared to be taking at baseline. Data is presented as n (%). Table 4 Summary of adherence based on attendance marked in trainers’ manuals and individual walkers’ logbooks   Walking N Mean±SD Walking & Behavioural N Mean±SD Self-directed (C) N Mean±SD Walking vs. Self-directed t -test P value Walking & Behavioural Vs. Self-directed t -test P value Walking & Behavioural Vs. Walking t -test P value 0-3 months 79 69 73 *(0.043) *(0.012) (0.514)   0.770±0.299 0.802±0.290 0.652±0.403       3-6 months 79 69 73 (0.242) (0.159) (0.774)   0.617±0.410 0.636±0.390 0.535±0.459       6-9 months 79 69 73 (0.421) (0.937) (0.363)   0.471±0.418 0.534±0.425 0.528±0.463       9-12 months 79 69 73 (0.549) (0.551) (0.989)   0.446±0.441 0.445±0.433 0.490±0.462       Total Adherence 79 69 73 (0.690) (0.413) (0.619)   0.576±0.346 0.604±0.342 0.551±0.420       Table 5 Summary of self-efficacy, measured with Stanford questionnaire on chronic disease for three study arms (continued)   Baseline 12 months 18 months   W WB C W WB C W WB C   N N N N N N N N N Variables Mean±SD Mean±SD Mean±SD Mean±SD Mean±SD Mean±SD Mean±SD Mean±SD Mean±SD         W.Vs. C(P) W.Vs. C(P) W.Vs. W(P) W.Vs. C(P) W.Vs. C(P) W.Vs. W(P) Coping 79 69 73 44 44 40 44 41 36 With 1.069±794 1.126±0.775 1.013±0.946 0.864±0.771 1.239±0.730 1.192±0.835 1.064±0.952 1.388±0.856 1.342±1.127 Symptoms       (0.091) (0.858) (0.057) (0.190) (0.793) (0.286) Confidence 79 69 73 44 44 39 43 41 36 About 7.406±1.719 7.827±1.488 7.858±1.512 7.826±1.551 7.682±1.785 8.064±1.575 7.690±1.920 7.546±1.848 8.015±1.476 Doing Things       (0.433) (0.297) (0.060) (0.235) (0.422) *(0.041) (Higher is better) W: Walking only group; WB: Walking and Behavioural Group; C: Self-directed group (unsupervised/self-directed); N: number of subjects in each comparative group; SD: standard deviation; vs: versus; data is presented as mean (standard deviation); p: p-value (statistical significance); * Statistically significant. BODY.METHODS.INTERVENTION.SUPERVISED WALKING PROGRAMS (WB & W GROUPS): The SCAWPs took place at two walking club sites in Ottawa, Ontario, Canada and one in Gatineau, Québec, Canada. Participants took part in three weekly walking sessions over a 12-month period. Every walking session began with a 10-minute warm-up, consisting of light aerobic exercises, before engaging in the 45-minute aerobic walking phase. The walking sessions ended with a 10-minute cool-down consisting of light aerobic exercises and stretching. The target intensity of the walking phase was approximately 50% to 70% of the subjects' pre-determined maximum heart rate as recommended in The Ottawa Panel guidelines [33-35]. The SCAWP was divided into two stages: 1) A "progressive aerobic phase" wherein the duration and heart rate intensity progressively increased over time and 2) a "maintenance aerobic phase" wherein the duration and heart rate intensity remained constant for the remainder of the walking program (Additional file 2). The selected dosage, frequency, intensity, and progression of the walking interventions were based on existing protocols proven effective for OA, described in several RCTs involving individuals with OA [33-35] (Additional file 2). To avoid contamination between the two walking interventions, participants in the W group were instructed to walk in the mornings, while the WB group walked in the afternoons at the main walking site. In order to meet the prescribed target heart rate, heart rate monitors were provided to each participant prior to his/her walking session. As a safety precaution, participants who experienced prolonged pain lasting more than two hours within 24 hours after a walking session were asked to temporarily reduce their level of exercise until he/she felt comfortable enough to resume the appropriate duration, intensity and frequency of the walking program. Following randomization, the PA specialist met each subject in the walking groups to explain the SCAWP and its progression (Additional file 2) and was present for a minimum of three weekly scheduled sessions over one year to supervise the subjects. The PA specialist was responsible for monitoring and recording attendance, blood pressure, heart rate (during and after the walking session), time duration, and number of steps (pedometer) at each walking session. Monetary compensation was given to each participant for attending each walking session. BODY.METHODS.INTERVENTION.BEHAVIOURAL INTERVENTION (WB GROUP): The behavioural intervention was part of the multifaceted KT intervention (Table 1) and was implemented using the adapted Program for Arthritis Control through Education and Exercise (PACEex) program [40]. The behavioural intervention was integrated into the PACEex program and consisted of the following components: (1) short- and long-term goal setting during PACEex classes; (2) an educational component, delivered by a trained instructor, involving consisting of instructional sessions about the benefits of PA, specifically walking; (3) monthly face-to-face counselling wherein participants received moral support/encouragement to adherence with PA. Potential barriers to program adherence were identified and self-management strategies were reviewed to overcome those barriers; (4) goal setting and telephone counselling were provided as an additional source of social support until the end of the supervised phase. As with the face-to-face counselling, barriers were identified and strategies were shared in an effort to promote the long-term maintenance of PA. In summary, the behavioural intervention consisted of twenty 2-hour group sessions discussing short-term goal setting and education of arthritis-related topics over a duration of 20 consecutive weeks. Individual long-term goal setting was discussed at the beginning of the PACEex program and was followed by monthly face-to-face meetings throughout the first 6 months of the program. The last six months of the 12-month supervised phase consisted of participants receiving counselling via telephone discussing long-term goals and barriers/facilitators to adhere to the walking program. BODY.METHODS.INTERVENTION.THE SELF-DIRECTED CONTROL (C GROUP): Participants were invited to consult an educational pamphlet on walking and OA. One introductory session was provided to the participant wherein they were provided an educational pamphlet describing the benefits of walking for OA, a pedometer to monitor PA, and a logbook to record activity level and adherence while partaking in the self-directed walking program. Participants in this group received monetary compensation for the completion his/her log books. The self-directed (C) group did not have any contact with participants in the two other groups, avoiding potential contamination. BODY.METHODS.DATA COLLECTION: Participants were assessed by an independent evaluator at baseline and at each 3 month interval (months 3, 6, 9, 12) during the intervention period. Participants were then assessed at 3 and 6 months post-intervention during the follow-up period (15 and 18-months). Participants were asked to complete a collection of validated questionnaires as well as perform physical tests at each assessment. BODY.RESULTS.KNOWLEDGE (CPG) UPTAKE OUTCOME MEASURES (KT OUTCOMES).ADHERENCE AND BEHAVIOUR CHANGE: Similar to other studies [29,41,42], participant adherence was assessed as the number of attended walking sessions divided by the number of prescribed sessions (3 sessions per week). Among the W and WB groups, the number of attended walking sessions throughout the 12-month duration was recorded by the exercise therapist on site as well as through the use of participants' completed logbooks which provided information on the amount of performed PA during the 12-month intervention period. The C group's adherence was only assessed by the completed log books. Behaviour change was based on the concept of self-efficacy and was measured using the Stanford scale. In regards to the research question, behaviour change results were only reported at 12 and 18-months. BODY.RESULTS.KNOWLEDGE (CPG) UPTAKE OUTCOME MEASURES (KT OUTCOMES).ANALYSIS: The analysis was conducted on an intention-to-treat basis. Descriptive statistics including mean, standard deviation and frequencies were used to summarize the study groups at baseline. A repeated measure mixed model was used to assess the change in adherence from baseline to end of treatment (12 months) among three study groups. Behavioural change outcomes were assessed from baseline to of end treatment (12 months) and at 6-month follow-up post-intervention (18 months). The model included variables such as intervention group, study month, and an interaction term between intervention group and study month. Missing data was assumed to be missing at random (MAR) in order to include incomplete data. Pairwise differences comparing each group to one another (W vs. C, W vs. WB, WB vs. C) were assessed. The repeated measure mixed model was used to compare the change of adherence from baseline to the 18- month follow-up. Statistical analyses were performed using SAS (version 9.2, SAS Institute Inc., Cary, North Carolina), and statistical significance was defined as p<0.05. BODY.RESULTS: Between January 2007 and December 2008, 629 study participants were assessed for eligibility. Figure 2 exhibits the flow of participants from recruitment to follow-up. The most common reasons for exclusion were: 1) time commitment (26.5%); 2) first diagnosed with OA by physician or X-Ray but later contradicted by MRI test result (18.1%); 3) loss of interest (14.2%); 4) severe OA of the weight bearing joint of LE (11.8%); 5) no medical clearance after VO2 max test (5.1%); 6) unable to contact (4.9%); 7) too active (2,9%); 8) knee replacement (2.7%); 9) age related (2.5%); 10) travelling too much (1.9%); 11) living too far (1.2%). Follow-up for the last recruited participant was completed in May 2010. Figure 2Study flow diagram. This figure provides a flow diagram of data collection from baseline to 18-months follow-up. The groups were similar in age, sex, ethnicity, OA duration and medication use (Table 2 &3). Two hundred and twenty two subjects were included in the study. Women made up 153 of the 222 subjects. The mean age (±S.D.) was 63.4 (±8.6) years, the mean disease duration (±S.D.) was 10.3 (±9.3) years, the highest level of completed education was "College graduate" with 65 participants (±29.3) and "Professional or graduate school" with 51 participants (±23.0). There was no statistical difference between the three comparative groups at baseline for all the demographic variables. BODY.RESULTS.BLINDING: The effectiveness of blinding the evaluator to treatment allocation was assessed through a questionnaire where she was asked to identify which group each participant belonged to. The blinding rate of the evaluator was high (98%). BODY.RESULTS.ADHERENCE AND ATTRITION: From baseline to 12 months, the adherence rates were expressed as percentages of adherence among walkers who did not drop out and were reported as means (+/−standard deviation) for each time period between groups (Table 4). Statistically significant results for short-term adherence rates favoured participants in the WB group (80.2%) compared to the self-directed (C) group (65.2%) (p<0.012) after 3 months. For long term adherence (6 to 12 months), the WB group demonstrated superior adherence rates throughout each period compared to the other two groups, but results did not reach statistical significance. No statistical significance (p> 0.05) was observed for the total adherence rates between WB vs. W, W vs. C, and WB vs. C. During the 12-month intervention period, the dropout rates were 43.1% for the W group, 40.6% for the WB group and 49.3% for the self-directed (C) group. From baseline to 18 months, the dropout rates were 44.3% for the W group; 40.6% for the WB group and 52.1% for the self-directed (C) group (Figure 2 and Additional files 3 &4). The dropout rates were lower for the WB group at 12-months (36.2%) and 18-months (39.1%) compared to the W group (40.5% and 44.3%) and the self-directed (C) group (40.5% and 51.3%). The W and self-directed (C) groups had the highest dropout rates (40.5%) at 12 months while the self-directed (C) group had the highest at 18 months (52.1%). BODY.RESULTS.BEHAVIOUR CHANGE: After the 12 month intervention phase, no differences were found between the 3 comparative groups for any of the items on the Stanford scale (Table 5). Moreover, variables related to coping with symptoms and confidence were higher among the WB group compared to the other two groups. At 18-months, statistically significant differences in scores between the WB and W groups revealed that the W group demonstrated an improved mean for the "confidence about doing things" variable (p=0.041). The self-directed group (C) demonstrated the highest mean "confidence about doing things" score 8.015± 1.476 (p=0.040) as it increased by 0.157 (p=0.048) when compared to baseline. The self-directed group (C) demonstrated the highest mean score 8.015± 1.476 (p=0.040). BODY.DISCUSSION.THE IMPACT OF THE MULTIFACETED KT INTERVENTIONS ON KNOWLEDGE (CPG) UPTAKE.ADHERENCE AND ATTRITION: The additional behavioural approach of the multifaceted KT intervention (WB group) improved adherence to the SCAWP when compared to the other two groups over a short-term period. In addition, the WB group demonstrated reduced dropout rates compared to the W and self-directed (C) groups. Over a long-term period, the three groups were equivalent in improving behaviour change while adopting the Ottawa Panel recommendations on walking [33-35]. Adherence rates were higher among the WB group for each 3-month period compared to the other two groups. Improved adherence rates over the short-term period can be explained by the fact the PACEex program used in the behavioural intervention lasted for 20 consecutive weeks at the beginning of the study. The PACEex program [40] used in this study, allowed our participants to set general goals, which may have motivated participants to adhere to the walking program. Similar to a previous study [43], another reason may be that participants in the W and WB group lost motivation after the supervised phase, no longer having regular professional support from the PA specialist. The drop-out rate results are comparable to other long-term studies which have used an aerobic PA intervention with a behavioural intervention component [38,44-54]. Higher adherence rates were demonstrated at 2 to 4 months (85% - 90%) and at 10 to 18 months (50% - 90%). An initial 6-month center-based exercise program enhanced retention over short and the long-term periods while promoting short-term adherence and energy expenditure [55]. The moderate exercise intervention retained more subjects, but had little influence on adherence over a long-term period [56]. Home-based PA programs, such as self-directed walking, can achieve improved adherence rates compared to facility-based programs over a long period [55]. BODY.DISCUSSION.BEHAVIOUR CHANGE: There were no statistically significant results among the three groups at 12 months. These results concur with a similar study [53] and are normal in the context of a KT study which implemented an already proven effective walking program [33-35]. However, at 18-months, there were statistically significant results which favoured the self-directed group (C). One reason explaining why participants in the self-directed group (C) had higher confidence and self-efficacy scores may be due to the fact that since these individuals were not supervised, they may have been more independent, self-controlled, self-confident, and self-motivated. Participants in this group may have also had the opportunity, over the 12-month period, to develop confidence and self-efficacy skills compared to the two supervised groups during the unsupervised follow-up phase. The variable "confidence about doing things" decreased after 12 months for the WB group. Given the high amount of PA demonstrated by the WB group at 12 months, we were surprised with this result. On the other hand, at 18 months, statistically significant results favoured the W group compared to WB group for the variables "confidence about doing things". Again, these results about confidence were unexpected given that the WB group performed higher amounts of PA. Exercise therapists, pedometers, logbooks and social support from walking club members (Table 1) assisted participants who had difficulties with activity adherence and maintenance [57]. Regular professional contact was recognised as the significant motivational factor for adherence to the supported walking program during the RCT [43]. Social support has been reported to be more successful in engaging participants in PA programs compared to programs which only provided written educational material [58,59]. The combination of using pedometers and goal-setting has been recognised as an effective KT tool for increasing PA [31,60-62]. However, despite the short-term effectiveness of pedometers and goal-setting, the success of these tools diminished over time. Other KT strategies are recommended to facilitate sustainability [62]. BODY.DISCUSSION.LIMITATIONS: Barriers related to the implementation of SCAWP were identified and addressed to improve adherence for older individuals [57]. Acceptability (preferences, tolerance and accessibility) should be considered to promote PA adherence and to overcome associated barriers [63]. The most common barriers identified by participants with OA are perceptions about illness and recovery, transportation difficulties, family commitments and inconvenient timing [64]. Time commitment is also recognised as a significant barrier [55]. Additional barriers such as weather conditions including snow storms, freezing rain and humidity, a 3-month bus strike, and health problems of participants' partners may have contributed to study adherence. Adherence is influenced by participant's preference of intervention [65-70]. Unfortunately, with this study being a RCT, taking participants' preference into consideration was not possible due to the randomization procedure. This being said, randomisation may have contributed to higher dropout rates. The purpose of this KT study was to implement a scientifically proven effective walking program for OA [33-35]. Short term research suggests that self-management interventions [16,18], telephone counselling [22,46,71-73], peer support [13,74-76] and PA education [18,22,27,41,77] are effective behavioural strategies for improving self-efficacy. The long-term multifaceted behavioural strategy used in this study did not concur with the previous studies, as participants in the self-directed group (C group) were provided information and education on the benefits of PA. Methods of measurement for adherence for the W and WB groups were different than the self-directed (C) group. Observed attendance was recorded on site for each walking session for the W and WB groups by exercise therapists while adherence could only be measured in the self-directed group (C) using completed logbooks. The self-reporting of PA, which is commonly overstated, may have been subject to potential information bias. Selection bias may have occurred as our study population included individuals with mild to moderate OA, with a confirmed X-ray report. In addition, some participants were retired while some were still employed. Since participants were provided monetary compensation for their participation in the study, one may argue the generalizability of the results. BODY.DISCUSSION.IMPLICATIONS: This KT RCT aimed to identify the best multifaceted strategy to implement a proven effective walking program for OA of the knee among older individuals. The epidemic public health problem of OA can be managed through the implementation of a proven effective walking program in existing community-based walking clubs as well as at home with minimal support. More studies are needed to improve the long-term walking adherence or knowledge uptake on SCAWP among participants with OA. Particular attention should focus on patients' characteristics and preferences. BODY.CONCLUSIONS: This study partially supported our initial hypothesis regarding the impact of a multifaceted KT intervention as the combined walking and behavioural approach had greater benefits over the comparative groups resulting in improved long-term adherence to PA only over a short period (3-months). The WB group demonstrated to be the best KT strategy for reducing dropouts compared to the W group and self-directed (C) group. All three KT strategies were equivalent over the long-term period (up to 18 months) for improving behavioural change. However, the self-directed walking program (C group) was the least expensive to implement over a long-period (18 months). This RCT was a long-term adherence study as well as a KT study which addressed questions of clinical and scientific importance aimed at improving the understanding of effective KT strategies to promote the adoption and maintenance of a community-based walking program for OA. BODY.ABBREVIATIONS: OA: Osteoarthritis; QoL: Quality of Life; SCAWP: Supervised Community-based Aerobic Walking Program; CPG: Clinical Practice Guideline; KT: Knowledge Translation; KTAC: Knowledge-To-Action Cycle; EBCPG: Evidence-Based Clinical Practice Guidelines; CONSORT: Consolidated Standards of Reporting Trials; WB (group): Walking and Behavioural intervention; W (group): Walking intervention; C (group): Self-directed control intervention; RCT: Randomized Controlled Trial; PA: Physical Activity; PACE-ex: Program for Arthritis Control through Education and Exercise; AIMS2: Arthritis Impact Measurement Scale 2; SF-36: Short-Form 36 Health Survey; WOMAC: Western Ontario and McMaster Universities Osteoarthritis Index; MET: Multiples of basal metabolic rate; MAR: Missing At Random; CIHR: Canadian Institutes of Health Research. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS’ CONTRIBUTIONS: LB is a Full Professor of Rehabilitation, an epidemiologist and the principal investigator of this study and primary author of this manuscript. GAW the co-principal investigator of this study, is senior biostatistician and director, Cardiovascular Research Methods Centre at the University of Ottawa Heart Institute, and is a leading expert in the design and analysis of clinical trials. He provided assistance with the methodology and statistical analysis of the study. GPK is a Full Professor of Physiology at the University of Ottawa, and director of the university's Human Performance and Environmental Medicine Research Laboratory and Professional Fitness and Lifestyle Consultant Certification Training program. He assisted with the methodology of the study. RR is a senior researcher at the University of Ottawa Heart Institute and provided experience in applying innovative behavioural approaches aimed at increasing PA in healthy and chronic diseased populations. AM is a health economist and assisted with the economic evaluation concept in the original proposal. PT is a rheumatologist, an epidemiologist and chief of the Cochrane Musculoskeletal Group. He has experience in conducting RCTs and meta-analyses. He provided assistance with OA outcome measures in the study. MH & CM developed the PACEex program and was in charge of training the PA specialist. GDA was the research coordinator of this study and assisted with the writing of this manuscript. LC is a biostatistician and performed the analyses of this study. All authors read and approved the final manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2458/12/871/prepub BODY.SUPPLEMENTARY MATERIAL: Additional file 1The knowledge –to-action framework and KT interventions. This table demonstrates how each step of the KTAC framework is applied to the RCT.Click here for file Additional file 2Post-Randomization Walking Interventions. The supervised phase of the walking intervention lasted 52 weeks following randomization. The unsupervised (follow-up) phase of the walking intervention lasted 26 additional weeks. For Intervention (one year): 52 weeks × 3 sessions =156 sessions attended; Follow-up (six month): 26 weeks x 3 sessions = 78 sessions attended. N.B. This structured and supervised walking program is based on Ottawa Panel Grade A recommendations [33-35] and was implemented to groups W and WB (Implementation groups). Individuals with OA in the self-directed group (C) received a pamphlet on OA and walking (which recommends regular walking in a unsupervised/self-directed way) (Dissemination group) [25].Click here for file Additional file 3Reasons of dropouts. This table demonstrates the reasons as to why participants decided to withdraw from the study.Click here for file Additional file 4Dropout rates and corresponding retention rates at 12-and 18-month time periods. This table demonstrates the drop-out rates and retention rates at end of intervention 12-months and follow-up at 18-months.Click here for file

TITLE: Treatment costs associated with interventional cancer clinical trials conducted at a single UK institution over 2 years (2009–2010) ABSTRACT.BACKGROUND:: The conduct of clinical trials should be an integral part of routine patient care. Treating patients in trials incurs additional costs over and above standard of care (SOC), but the extent of the cost burden is not known. We undertook a retrospective cost attribution analysis to quantitate the treatment costs associated with cancer clinical trial protocols conducted over a 2 year period. ABSTRACT.METHODS:: All patients entered into oncology (non-haematology) clinical trials involving investigational medicinal products in 2009 and 2010 in a single UK institution were identified. The trial protocols on which they were treated were analysed to identify the treatment costs for the experimental arm(s) of the trial and the equivalent SOC had the patient not been entered in the trial. The treatment cost difference was calculated by subtracting the experimental treatment cost from SOC cost. For randomised trials, an average treatment cost was estimated by taking into account the number of arms and randomisation ratio. An estimate of the annual treatment costs was calculated. ABSTRACT.RESULTS:: A total of 357 adult oncology patients were treated on 53 different trial protocols: 40 phase III, 2 randomised II/III and 11 phase II design. A total of 27 trials were academic, non-commercial sponsored trials and 26 were commercial sponsored trials. When compared with SOC, the average treatment cost per patient was an excess of £431 for a non-commercial trial (range £6393 excess to £6005 saving) and a saving of £9294 for a commercial trial (range £0 to £71 480). There was an overall treatment cost saving of £388 719 in 2009 and £496 556 in 2010, largely attributable to pharmaceutical company provision of free drug supplies. ABSTRACT.CONCLUSION:: On an average, non-commercial trial protocols were associated with a small per patient excess treatment cost, whereas commercial trials were associated with a substantially higher cost saving. Taking into account the total number of patients recruited annually, treatment of patients on clinical trial protocols was associated with considerable cost savings across both the non-commercial and commercial portfolio. BODY: The drug development process is well established and randomised clinical trials remain the gold standard for testing new treatments against standard of care in order to advance clinical practice (Vogelzang et al, 2012). Even so, the proportion of patients entering clinical trials worldwide is remarkably low. Since 2001, when under 4% of cancer patients took part in a clinical trial, recruitment increased six-fold over the following 10 years in the UK due to significant investment in a unique national research infrastructure (Department of Health, 2006; NCRN, 2011), whereas recruitment in the USA has remained static (Califf et al, 2012). It is undisputed that conducting research incurs additional costs over and above standard of care (Emanuel et al, 2003), yet there is an international commitment to research being a core health service function (Zerhouni, 2003; Califf et al, 2012; The NHS Constitution, 2013). Attribution of research costs is complex and recovery of those costs, particularly for non-commercial clinical trials is very challenging (Snowdon et al, 2006; Seow et al, 2012). Costs incurred by conducting a clinical trial can broadly be attributed to one of the three elements (Attributing the costs of health and social care Research & Development (AcoRD), 2012): (1) research costs, that is, the costs of the trial itself, which end when the research ends and relate to activities being undertaken to answer the research question; (2) treatment costs, that is, the patient care costs, which would continue to be incurred if the patient care intervention continued to be provided after the trial had stopped; and (3) service support costs, that is, the additional patient care costs associated with the research, which would end once the trial had stopped, even if the patient care involved continued to be provided. For commercially sponsored trials, all three elements would normally be met by the sponsor. In the UK, pharmaceutical companies have, for many years, been able to access service support from the national health service by formal adoption into a national clinical trials portfolio. For non-commercial studies, research costs are generally met by grant funders. Service support costs are met from local R&D budgets (via comprehensive local research networks), whereas treatment costs are met through the normal commissioning process. The latter has been the cause of significant disharmony between commissioners and providers of health care. In the USA, private insurance companies are not obliged to cover non-routine patient care costs (Anonymous, 2007). Cancer drugs are generally expensive and it is widely perceived that cancer clinical trials generate an excess treatment cost, despite a lack of evidence to support this view. A single retrospective review of 932 cancer patients recruited to 35 trials in the USA between 1998–1999 suggested that the incremental costs of direct care in clinical trials was 6.5% higher than what they would be outside of a trial (Goldman et al, 2003). The investigators concluded that the additional treatment costs of an open reimbursement policy for government-sponsored cancer clinical trials seem minimal. Even so, risk of excess treatment costs remains a barrier to academic clinical trials being performed in many countries today. We undertook a review of cancer clinical trials conducted in a single, research-active institution in the UK over a 2-year period (2009–2010), to identify the extent of treatment costs involved. BODY.MATERIALS AND METHODS: Cambridge University Hospitals NHS Foundation Trust (Addenbrooke's Hospital, Cambridge) is a major university teaching hospital and cancer centre in the UK. It also hosts the West Anglia Cancer Research Network (WACRN), one of the highest recruiting regional research networks in the UK, where 45.1% of patients newly diagnosed with cancer entered a research study in 2011/12. Although WACRN supports research across seven hospitals in the region, 64% of all patients were recruited at Addenbrooke's hospital. For this retrospective cost attribution analysis, all patients recruited to interventional, research ethics committee approved, oncology drug trials between 1 January 2009 and 31 December 2010 at Addenbrooke's Hospital were identified from the Cambridge Cancer Trials Centre patient database. Haematology trials were excluded. The clinical trial protocols for each identified patient were reviewed to determine the treatment costs associated with each arm of the trial. The following activities were attributed to being treatment costs (as described in the AcoRD guidance): (1) supplying an investigation medicinal product and pharmacy costs associated with administering oral agents under investigation; (2) supplying and administering any active comparators, but not placebo; (3) nursing time to administer the drug, time taken to deliver the treatment either as an outpatient ('day unit chair time') or as an inpatient ('bed days'); (4) investigations, including imaging and laboratory tests, which would continue to be incurred if the patient care service in question continued to be provided after the trial stopped; (5) additional clinic visits. Costs were calculated for the active treatment phase of the trial only and did not include follow-up once treatment had finished. For all protocols, a standard of care (SOC) was identified as the standard treatment that would have been offered locally to the patient had the trial not been available. For randomised trials, if the control arm differed to the local standard treatment, the local standard treatment was used to cost SOC. The treatment costs of the SOC were determined as for the experimental arm. Costs were determined for the duration of treatment defined in each trial protocol and did not include costs of follow-up once treatment stopped. For adjuvant, neo-adjuvant and some palliative protocols which specified the treatment programme duration, costs were determined for the entire prespecified treatment programme. For palliative protocols where treatment duration was until disease progression, the actual number of cycles and/or months of treatment delivered to the patients recruited to each protocol was obtained from the patient records and an average treatment duration was determined for each protocol. The same treatment duration was used for estimating SOC costs, unless the SOC treatment maximum duration was specified, in which case the shorter duration was used. Cancer drug costs were obtained from the British National Formulary (2010) and excluded additional taxes or local negotiated discounts. Supportive drugs such as antiemetics, antidiarrhoeals, steroids, intravenous hydration fluids were excluded. For dose calculations, an average body surface area of 1.75 m2 was used. Staff and facility direct costs without overheads were obtained from local reference costs. Imaging and laboratory tests were costed locally or using the NIHR costing template. Where costs differed, local costs were used. The data was cross-checked with the lead clinical trials nurse responsible for each protocol. For each protocol, the treatment cost (TC) difference for the experimental arm(s) was the difference between the experimental arm treatment cost and the SOC cost. For randomised trials, an average treatment cost was attributed to the protocol, by taking into account the number of arms of the trial and randomisation ratio (see Table 1 as an example of how the TC difference was calculated). Finally, using the total number of patients recruited to each trial protocol, the annual overall TC excess or saving in 2009 and 2010 was estimated. BODY.RESULTS: During the 2-year review period, 357 cancer patients were recruited to 53 different interventional clinical trials involving IMPs (Tables 2 and 3): 159 patients in 2009 and 198 patients in 2010. A total of 27 trials were academic, non-commercial sponsored trials with variable levels of industry support. A total of 26 trials were commercial sponsored trials, of which 26 of the non-commercial trials and 9 of the commercial trials were adopted in the NIHR portfolio. Thirty-nine of the 53 trials were randomised phase III design. The remainder were 1 non-randomised phase III, 2 randomised phase II/III, 9 randomised phase II and 2 non-randomised phase II trials. Patients received IMPs that were either unlicensed (22), licensed for the indicated disease (10) or licensed drugs being used in an unlicensed condition (21). Compared with 2009, in 2010, 24 new trials had opened to recruitment and 12 had closed. The average TC per protocol across the portfolio of 53 trials ranged from an excess of £6393 to a saving of £71 480 (Figure 1). For 27 non-commercial trials (Table 2), the average TC per protocol ranged from an excess of £6393 to a saving of £6005 (mean excess TC was £431, median £126 per protocol). The main source of excess TC was due to extra hospital day unit delivery time associated with 9 protocols evaluating IMPs given in addition to, or in place of, SOC (7 protocols) or where the SOC was observation (2 protocols). For example, in the AVAST-M clinical trial comparing 12 months of 3 weekly adjuvant bevacizumab as treatment for patients with resected melanoma versus standard observation, the additional cost of treatment delivery accounted for £4250 (83%) of the £5144 excess TC. In this case, the investigation medicinal product cost did not contribute to the excess TC, as all bevacizumab was supplied free of charge by the manufacturer. A total of 7 non-commercial trials were associated with a TC saving. Two of these trials, SCOT (comparing 3 months versus 6 months standard adjuvant chemotherapy in colorectal cancer) and PERSEPHONE (comparing 6 months versus 12 months standard adjuvant chemotherapy in breast cancer), were evaluating whether shorter durations of adjuvant treatment were as effective as SOC, in which the TCs were, in effect, halved. In 2 trials, the source of saving was free drug supplies in the experimental arms from manufacturers (SoFEA and BR13). As each trial randomised one or more treatment against a standard treatment, taking into account the number of arms and randomisation ratio, the average cost of a patient recruited to the protocol was less than SOC. In one trial (REAL-3), an industry grant provided a per payment fee of £1000 and in another trial (Telovac), one of the experimental arms was associated with fewer cycles of SOC chemotherapy given in association with the investigation medicinal product. In one trial (QUASAR2) comparing adjuvant capecitabine with capecitabine plus bevacizumab, the manufacturer supplied both the investigation medicinal product (bevacizumab) and the SOC (capecitabine) free of charge for patients recruited to either arm of the trial. With the exception of REAL-3, treatment of patients on the other six non-commercial trial protocols associated with an overall TC saving offered financial benefits that would have been passed on to commissioners, primarily by the provision of free drug that would have had to be paid for if the patient was not on trial. Considering the actual numbers of patients recruited to all of the non-commercial trials annually, the net TC saving was estimated at £67 962 in 2009 and £29 002 in 2010. For the 26 commercial trials (Table 3), the average TC per protocol ranged from £0 to a saving of £71 480 (mean saving was £9294, median £4370 per protocol). Commercial trials were not associated with excess TCs, as income from the trial conduct was gained to cover all costs associated with treatment. The main source of cost saving was through provision of free drug supplies from the sponsor. In two renal cancer trials (Bevlin and Rad001), the sponsors provided the experimental drug free and in a third renal trial (Torisel404) both experimental and SOC drug were provided. With the exception of four randomised trials where SOC was observation or best supportive care, treating patients on the remaining 22 protocols would have passed on significant drug cost savings to commissioners, by avoiding the SOC costs the commissioners would have incurred if the patients were not on trial. Using the actual numbers of patients recruited to all of the commercial trials, there was an estimated net treatment cost saving of £320 758 in 2009 and £467 554 in 2010. Figure 2 summarises the distribution of average TCs for all 26 non-commercial and 27 commercial trial protocols. In general, it can be seen that both commercial and non-commercial trials saved drug costs, whereas the extent of drug and dispensing cost savings was markedly higher for commercial compared with non-commercial trials. The main source of excess TC was the cost of delivering treatment, most often generated by trials evaluating IMPs given in addition to SOC. The contribution of extra clinic visits and investigations, in particular CT scans used for response assessment, was significantly higher for commercial compared with non-commercial trials. By taking into account the number of patients entered on each protocol, the overall annual TCs incurred were estimated. On an average, treating a patient on a clinical trial protocol was associated with a TC saving of £4340. The overall TCs incurred annually was a saving of £388 719 in 2009 and £496 556 in 2010 (Figure 3), around 90% of the savings coming from commercial sponsored trials. BODY.DISCUSSION: Research is considered an integral part of healthcare by many nations, as the benefits of new treatments tested in clinical trials are recognised to improve patient outcomes. It is important that the cost of that research is identified and properly funded. In the UK, the AcoRD guidance (2012) provides a framework to identify, attribute and recover the various costs associated with research, in a transparent, robust and consistent manner. It clarifies the distinction between research costs, service support costs and TCs. We have focused on quantitating the TCs associated with treating patients in cancer clinical trials, as uncertainty surrounding the size of cost difference between treatment in or outside of trials, particularly non-commercial trials, is a barrier to their efficient conduct. We have previously sought to estimate the cost of the treatment administered to patients recruited to cancer trials using individual patient data from clinical research forms and pharmacy prescriptions (Corrie et al, 2011). However, TCs extend beyond the drug costs alone. So, to better inform service delivery, we developed a methodology designed to include all elements of TCs as described in the AcoRD guidelines. The method provides a unit TC difference associated with an individual trial protocol, taking into account the number of arms in a trial and, where appropriate, the randomisation ratio. The unit TC difference can only be an estimate, as the duration of treatment may vary for an individual patient, whereas for randomised trials, one might expect the estimate to be more accurate the more patients recruited to any protocol. In addition, we tailored SOC to local treatment guidelines, which may differ between regions. There are several limitations associated with this methodology. First, we did not take into account changes to the treatment plan associated with adverse drug reactions and did not include any associated non-elective hospital admissions, as individual patient data was not analysed in depth other than to estimate duration of palliative treatment in some instances. In addition, we did not include follow-up of patients after completion of anticancer therapy, assuming that this would account for a proportionally small element of treatment costs: commercial trials would be expected to fully reimburse all costs above SOC, whereas in general, frequency of follow-up in non-commercial trials tends to reflect routine follow-up arrangements. Finally, costing of commercial trials was based on direct costs only and did not include overheads charged to industry. Despite these limitations, we believe this methodology has the potential to be applied on a wider scale to inform providers and commissioners regarding the impact of conducting cancer clinical trials on health service resources. Review of patients recruited to interventional cancer trials in a single research active institution over a 2-year period identified significant variation in associated TCs between protocols. The average TC for 27 non-commercial trials was an excess of £431 (range £6393 excess to £6005 saving) and for 26 commercial trials was an average saving of £9294 (range £0 to £71 480). Breaking down the TCs by the main categories, by far the largest contribution to cost savings was provision or reimbursement of IMPs by pharmaceutical companies, primarily as part of the conduct of commercial sponsored studies, but also, and to a lesser extent, in partnership with academic groups conducting non-commercial NIHR portfolio studies. The distribution of TCs was rather different for commercial and non-commercial trials. For non-commercial trials, the main excess TC was due to administering IMPs. For commercial trials, considerable extra costs were incurred by additional clinic visits and imaging investigations. This might suggest that pharmaceutical companies drive a more intensive patient management plan, and/or that, as many of the commercial protocols were for international multi-centre trials, they were designed to reflect practice in countries outside of the UK. Even so, because commercial trials were fully funded, the potential excess TCs associated with these protocols should not be passed on to commissioners. Our findings confirmed that the pathway for patients entered into academic trials did, in general, reflect routine clinical practice. By assigning the 357 patients to the protocols in which they were enroled, the overall treatment cost saving to the NHS was estimated to be approaching £0.5 million per annum. In our view, this data provides overwhelming evidence to refute any concern that clinical research generates a cost pressure for the health service. On the contrary, we have demonstrated significant financial gains. The largest gains were from commercial sponsored trials. There is currently a growing emphasis on expanding the commercial trial portfolio across all disease areas. Although this may bring financial benefits, the main reason for our undertaking this review was to address concerns that academic clinical trials were a financial liability. Taken individually, this may be the case in some instances, but viewed across a whole portfolio, there are clear gains even for non-commercial clinical trials. Academic trials represent an important part of the NIHR portfolio, affording the opportunity to address questions that would not be a priority for industry sponsors. For example, two non-commercial trials testing whether shorter durations of adjuvant therapy might be as effective as longer durations were associated with significant TC savings during their conduct, whereas positive outcomes would have much wider implications for health service savings. In summary, this unique analysis of TCs associated with patients recruited to cancer clinical trials indicates a wide range of per patient TC differences associated with individual trials compared with SOC, whereas the risk of there being an excess TC (as opposed to a saving) primarily lies with non-commercial studies. However, over the course of a year, clinical trials—both commercial and non-commercial—offer an opportunity to realise considerable TC savings. These findings support the Department of Health's directive for commissioning of clinical trials as an integral part of service delivery. A balanced portfolio of both commercial and non-commercial research should offer the greatest benefits to patients and the overall health economy.

TITLE: The effect of aerobic exercise in the maintenance treatment of depression ABSTRACT: We investigated the efficacy of aerobic exercise alongside antidepressant medication as an adjuvant maintenance treatment for depression. Fifty patients in remission were randomly assigned to either medication only or medication plus exercise. Assessment of psychopathology was made at 6-weekly intervals (for 24 weeks) using the Hamilton Rating Scale for Depression. The medication-plus-exercise group showed significantly more improvement at 12 and 24 weeks than the medication-only group. This study adds to a growing evidence base that suggests aerobic exercise is worthy of further development in the treatment of depressive disorders. BODY: The role of exercise in treating depression has been studied extensively in recent years. For example, Rethorst et al (2009) carried out a meta-analysis of 58 randomised controlled trials, and found that groups whose treatment incorporated exercise had significantly lower depression scores than controls. De Zeeuw et al (2010) showed that exercise could have preventive potential, as it was found to reduce the likelihood of depression in high-risk employees with sedentary jobs and an inactive lifestyle. Exercise has also been reported to reduce relapse rates. For example, Babyak et al (2000) found that participants with major depressive disorder who used exercise therapy alone had significantly lower relapse rates than those who received medication. Despite these promising findings and its low cost, the effectiveness of exercise is yet to be fully addressed in the psychosocial treatment literature or incorporated into mainstream treatment practice. For example, the National Institute for Health and Clinical Excellence (NICE, 2009) recommended 'structured group physical activity' only as a low-intensity psychosocial intervention for subthreshold depressive symptoms, or depression with chronic physical health problems. This may be because many studies have been criticised for their methodological limitations and incomplete literature searches (Rethorst et al, 2009); indeed, the classification and duration of exercise are not specified in the majority of reports. Of further note is that the efficacy of exercise in a maintenance capacity remains largely unexplored. Thus, our rationale was to investigate the role of exercise alongside antidepressant medication as a post-remission maintenance therapy, in terms of reducing both the severity of psychopathology and relapse rates. BODY.METHOD: The 50 patients recruited were attending the psychiatric out-patient department of Sir Sunderland Hospital, Banaras Hindu University, India. Selection was made (via referrals from two senior psychiatrists at the hospital) by the researcher who carried out the post-treatment evaluations. The following selection criteria were used: age 15–45 years, in order to cope with the physical exercise regimea diagnosis of major depressive or dysthymic disorder, according to DSM-IV-TR criteria (American Psychiatric Association, 2000)absence of any comorbid psychiatric illnessno major cardiac or orthopaedic illness that would contraindicate exercise. Ethical approval was granted by the University's research ethics committee. After inclusion in the study, patients were prescribed sertraline (200 mg/day – the maximum permitted daily dose) and alprazolam (1 mg/day) for 3–4 weeks in order to achieve remission of clinical depression. Once significant remission – defined as scoring less than 16 on the 21-item Hamilton Rating Scale for Depression (HRSD; Hamilton, 1960) – was obtained, patients were randomly allocated to groups; blinding was not possible, however, as participants were necessarily aware of the treatment group they were in and the researcher who administered exercise therapy also did the follow-up assessment of psychopathology. BODY.METHOD.PRE-TREATMENT EVALUATION: The researcher assessed the following using a semi-structured pro forma: sociodemographic and clinical dataseverity of depressive psychopathology (using the HRSD, plus a structured interview guide)stressful life events (lifetime and past 3 months), using the 51-item Presumptive Stressful Life Events Scale (PSLES; Singh et al, 1984) (a culturally appropriate instrument)melancholic and atypical features of depression (as per DSM-IV-TR)baseline heart rate (mean taken from three 1-minute time slots)fitness levels using the Harvard Step Test, to monitor compliance with the exercise programme (Brouha, 1943). BODY.METHOD.TREATMENT: Both groups continued to receive sertraline (200 mg/day) and alprazolam (1 mg/day) and did not take part in any form of counselling or psychotherapy. For the medication-plus-exercise group, participants also received training in daily exercise therapy. That therapy consisted of a 5-minute warm-up, 15 minutes of stationary running, and a 10-minute cool-down, this being convenient and economical for all participants regardless of gender or location and within guidelines on exer cise (Whaley et al, 2006). Participants were also advised not to perform any other exercise beyond usual daily activities. Heart rate recordings were made in order to adjust the intensity of exercise to keep within the target heart rate zone, this being 60–84% of the maximum achievable heart rate (Whaley et al, 2006); exercise sessions at home were logged and monitored by carers, and the logs were reviewed by the researcher at every follow-up. None of the participants or their carers reported missing any exercise sessions or cutting sessions short. Follow-up was at 6-weekly intervals for 24 weeks for all participants, and HRSD and PSLES scores were assessed on each occasion. The medication-plus-exercise group also received refresher exercise training and the Harvard Step Test was performed at every follow-up. BODY.METHOD.ANALYSIS: Statistical tests used were the chi-square and Student t-test. Epi Info 6 software, version 6.04a, was used to analyse the data. BODY.RESULTS: Although all 50 patients initially agreed to participate, seven did not complete the study (three from the medication-plus-exercise group and four from the medication-only group). Table 1 shows the sample characteristics and features of depression, which were evenly distributed between the groups. Table 2 shows the inter-group comparisons at all 6-weekly follow-ups. The medication-plus-exercise group showed a greater improvement in HRSD scores (change in score from baseline) than the medication-only group from the first follow-up, at 6 weeks, and this difference became statistically significant at weeks 12 (P = 0.04, t = 2.10) and 24 (P = 0.02, t = 2.42); there was no significant difference between the groups' PSLES scores at any follow-up point. Table 1 Baseline characteristics of the sample Variable Medication-only group ( n = 25) Medication-and-exercise group ( n = 25) Number (%) female 9 (60) 6 (40) Number (%) male 16 (46) 19 (54) Usual degree of physical activity: number (%)  sedentary  light  moderate 15 (65) 9 (41) 1 (20) 8 (35) 13 (59) 4 (80) Mean (s.d.) age (years) 29.6 (8.2) 30.5 (8.9) Mean (s.d.) PSLES score for lifetime 216.2 (124.9) 188.0 (86.3) Mean (s.d.) PSLES score for last 3 months 105.0 (72.6) 107.2 (65.3) Mean (s.d.) HRSD score * 23.2 (4.2) 22.8 (3.7) Mean (s.d.) number of melancholic features * 1.2 (1.6) 0.7 (1.1) Mean (s.d.) number of atypical features * 0.1 (0.33) 0.0 (0.3) HRSD, Hamilton Rating Scale for Depression; PSLES, Presumptive Stressful Life Events Scale. * Between group comparisons were non-significant. Table 2 Mean (s.d.) improvement (difference from baseline) in score on the Hamilton Rating Scale for Depression Follow-up Medication-only group Medication-and-exercise group t d.f. P 6 weeks 2.6 (5.3) 4.1 (3.4) 1.17 46 >0.05 12 weeks 2.3 (5.2) 5.0 (3.4) 2.10 45 0.04 18 weeks 3.0 (6.5) 5.6 (5.1) 1.45 42 >0.05 24 weeks 1.0 (7.0) 5.6 (5.4) 2.42 41 0.02 Relapse rates were also recorded via subjective reports and as a measured increase of >50% from baseline HRSD scores (Neumeister et al, 1997). In total, nine patients relapsed, two from the medication-plus-exercise group (both in week 18) and seven from the medication-only group (one relapse in week 6, two in week 12 and four in week 24). This difference in relapse rates did not reach significance (Fisher's exact test: one-tailed, P = 0.07; two-tailed, P = 0.14). BODY.DISCUSSION: We found that the maintenance treatment of medication-plus-exercise resulted in significantly lower HRSD scores than medication alone at 12-week and 24-week assessments, as well as a promising reduction of relapse rates. These differences were not considered to be due to extraneous life events, as there was no significant difference between the two groups' PSLES scores. These findings are in line with Rethorst et al's (2009) meta-analysis in terms of the potential for exercise to lower depression scores, and with the finding from Babyak et al (2000) that exercise is effective in reducing relapse. We found no studies, however, on the combined role of exercise and antidepressant medication as a maintenance therapy for depressive disorder. The present findings suggest a potential role for exercise as a protective agent during remission, and one which might reduce long-term cost. The study was limited in several ways. There is potential for bias via use of a clinician-rated tool such as the HRSD, particularly in an unblinded study. However, to reduce this, the same experienced assessor was used for the pre-intervention and follow-up assessments and a second assessor made random cross-checks at least once for every participant. Assessors completed the rating scales with the participants through discussion. The sample size was limited to 50, which precluded analysis of potential confounds. For example, the medication-only group was randomly allocated a greater proportion of sedentary members from the outset. There was also an unequal proportion of male/female participants; however, this reflected the population of patients within the hospital. A further limitation was the lack of comparison with an exercise-only group. It is possible that the exercise group may have derived social gain from the increased contact with carers and researchers; interestingly, Babyak et al (2000) raise the point that psychological factors such as proactive agency may be engaged via choosing to exercise. A further issue for the current study was the drop in the rate of improvement for the medication-only group at week 24. The reasons for this are unclear; however, such a change dictates that studies with longer periods of assessment are needed to make stronger interpretations. Thus, future studies could benefit from: larger and more representative samples; extended follow-up periods; additional outcome measures (including patient-rated scales); and the inclusion of further control groups, such as exercise only, social contact only, and group versus solitary exercise. In conclusion, this study adds to the growing evi dence base that suggests exercise is valuable in the treatment of depressive disorders. In particular, we highlight its potential role as a supplementary maintenance treatment during remission. It is worth highlighting that, despite the relatively short time for which the participants exercised, there were measurable benefits. We believe this area of research warrants further attention, particularly as exercise provides a low-cost, flexible and easily implemented treatment with minimal adverse effects, and especially where costly delays in receiving mainstream psychological interventions are commonplace.

TITLE: Singing teaching as a therapy for chronic respiratory disease - a randomised controlled trial and qualitative evaluation ABSTRACT.BACKGROUND: Despite optimal pharmacological therapy and pulmonary rehabilitation, patients with COPD continue to be breathless. There is a need to develop additional strategies to alleviate symptoms. Learning to sing requires control of breathing and posture and might have benefits that translate into daily life. ABSTRACT.METHODS: To test this hypothesis we performed a randomised controlled trial, comparing a six week course of twice weekly singing classes to usual care, in 28 COPD patients. The experience of singing was assessed in a qualitative fashion, through interviews with a psychologist. In addition, we surveyed patients with chronic respiratory conditions who participated in a series of open singing workshops. ABSTRACT.RESULTS: In the RCT, the physical component score of the SF36 improved in the singers (n = 15) compared to the controls (n = 13); +7.5(14.6) vs. -3.8(8.4) p = 0.02. Singers also had a significant fall in HAD anxiety score; -1.1(2.7) vs. +0.8(1.7) p = 0.03. Singing did not improve single breath counting, breath hold time or shuttle walk distance. In the qualitative element, 8 patients from the singing group were interviewed. Positive effects on physical sensation, general well-being, community/social support and achievement/efficacy emerged as common themes. 150 participants in open workshops completed a questionnaire. 96% rated the workshops as "very enjoyable" and 98% thought the workshop had taught them something about breathing in a different way. 81% of attendees felt a "marked physical difference" after the workshop. ABSTRACT.CONCLUSION: Singing classes can improve quality of life measures and anxiety and are viewed as a very positive experience by patients with respiratory disease; no adverse consequences of participation were observed. ABSTRACT.TRIAL REGISTRATION: Current Controlled Trials - ISRCTN17544114. BODY.BACKGROUND: Chronic obstructive pulmonary disease (COPD) causes breathlessness, which can occur on exertion or, in some individuals, at rest. Although therapy including bronchodilators, pulmonary rehabilitation and oxygen can improve symptoms, the underlying pathology is largely irreversible, meaning that many patients remain limited despite optimal therapy [1,2]. The pattern of breathing typically adopted in COPD is caused by the presence of expiratory flow limitation which leads patients to adopt higher operating lung volumes - dynamic hyperinflation [3]. Although this pattern allows increased expiratory flow rates, this comes at the expense of a greater inspiratory load and mechanical disadvantage for the respiratory muscles. A rapid breathing pattern reduces expiratory time and will exacerbate this. The disparity between respiratory work and ventilatory output that develops is an important determinant of symptoms [4]. A range of non-pharmacological measures for breathlessness have been trialled in respiratory patients [5], including pursed lip breathing [6,7], yoga [8,9], singing [10] and laughter [11], all based on the premise that changing patients' pattern of breathing will improve symptoms A limitation of some physiotherapy approaches is that by focusing attention on breathing pattern they may accentuate awareness of respiratory limitation [12]. Singing requires the development of skills in controlling posture and breath that might be transferrable to everyday life. Breathlessness is a complex sensation [13]; singing lessons might offer techniques that address both the sensory component - largely control of respiratory pattern to reduce hyperinflation, and the affective component - a conscious experience of using the breath for 'something positive'. In addition, there is some evidence that singing may have beneficial effects on wellbeing in healthy [14] and chronic disease populations [15,16]. To evaluate this further we used three approaches; firstly a randomized controlled trial of a six week course of singing training in COPD, secondly an evaluation of a program of open 'drop-in' singing lessons for patients with respiratory disease and thirdly a qualitative evaluation of patients' experience of participating in singing. BODY.METHODS.RANDOMIZED CONTROLLED TRIAL: Patients with COPD, diagnosed according to the GOLD guidelines, who were attending respiratory clinics at the Royal Brompton Hospital, were invited to participate. The study was approved by the Royal Brompton, Harefield and National Heart and Lung Institute Research Ethics Committee and all subjects gave written informed consent. The trial was registered at Current Controlled Trials - ISRCTN17544114. Demographics, height, weight, clinical history and spirometry (Microlab, CareFusion, Kent, UK,) were recorded. Participants completed the Hospital Anxiety and Depression (HAD) Questionnaire [17], St George's Respiratory Questionnaire [18], and the Short Form 36 Questionnaire [19]. Functional exercise capacity was assessed using the incremental shuttle walk test (ISWT). Time to recovery of oxygen saturation, Borg dyspnoea score and heart rate following the walk was documented [20]. Control of breathing was assessed using two measures. 1) Breath hold test - subjects held their breath from maximum inspiration [21,22]. 2) Single breath counting - subjects were instructed to breathe in and then count out loud in time with a metronome running at 60 beats/min[23]. Three attempts at each manoeuvre were made and the mean values recorded. Both techniques were in routine use in the physiotherapy department for the assessment of hyperventilation. All subjects received a thirty minute standard session on breathing control and techniques to manage breathlessness, delivered by one of two senior respiratory physiotherapists involved in the study. Pursed lip breathing and nose breathing were also discussed in relation to managing episodes of shortness of breath. Each subject received a standard Royal Brompton Hospital "Help Yourself - physiotherapy for people with respiratory symptoms" booklet (Additional file 1) and was advised to practice the techniques at home. At the end of the baseline session, patients were randomised to either the singing or control groups, using block randomization through consecutive sealed envelopes. The subjects in the singing group attended a hospital-based workshop, led by a singing teacher (PC), twice weekly for six weeks. Each session lasted approximately one hour and included teaching of posture, relaxation and vocal exercises. Tasks of increasing difficulty were given as the sessions progressed - described in more detail in additional file 2. Each subject was given homework and an accompanying CD of songs to practice at home. The teacher was unaware of the tests being used to assess breathing control, so that she would not 'teach to the test'. The control group had no further intervention. At seven weeks follow-up, study participants were again assessed by the same respiratory physiotherapists, who were blinded to treatment allocation. All subjects were instructed not to tell the physiotherapist which group they had been allocated to. Control of breathing, quality of life and functional exercise capacity were measured as at baseline. Statistical analysis was performed using StatView 4.0, using t-tests to compare change in outcome measures between patients and controls. A p value of < 0.5 was taken as significant. BODY.METHODS.EVALUATION OF OPEN SINGING WORKSHOPS FOR RESPIRATORY PATIENTS: Alongside the randomized control study, we ran open, twice-weekly, 'Singing for BreathingTM' workshops for patients with any respiratory condition who were at Royal Brompton Hospital either as inpatients, day cases or outpatients. Conditions included interstitial lung disease, cystic fibrosis, asthma, bronchiectasis and COPD. The sessions were advertised through posters in the hospital and by word of mouth to patients from the arts team, respiratory nurses, physiotherapists and other healthcare professionals. Patients involved in the randomized control study were excluded from these sessions as were those where infection control issues existed. The open sessions were led by the same singing teacher as the randomised study and encompassed relaxation and posture education, as well as phonation exercises. Patients attending these workshops for the first time were asked to complete an anonymous satisfaction questionnaire at the end (Additional file 3). BODY.METHODS.QUALITATIVE SURVEY OF PATIENT EXPERIENCE OF SINGING: Following completion of the randomized control study, a chartered counselling psychologist (AE), who had not otherwise participated in the study, interviewed a random sample of eight individuals who had been in the singing arm of the trial. The thirty minute interviews were structured and each subject was asked about 1) physical changes they had noticed. 2) Emotional changes they had noticed. 3) Behavioural changes they had noticed. 4) Any detrimental effects or negative experiences. Participants were assured that they could make negative comments if they wished and that their responses would be anonymous. The interview sheet is shown in additional file 4. BODY.RESULTS.RANDOMIZED CONTROLLED TRIAL: Participation in the study is described in the CONSORT diagram (Figure 1). Ninety two patients were approached to participate in the study. Fifty four did not wish to take part, one person who was already enrolled in another trial was excluded and one lived too far away to participate. Of the remaining thirty six patients, twenty were allocated to the singing group and sixteen to the control group. Of the singing group, two subjects withdrew from the study once the workshops had begun and three did not attend the final assessment once completing the workshops. In the control group, one withdrew and two did not attend the final assessment. Data are therefore presented for the 15 singers and 13 controls who completed the study. Of these, 6 attended 12 sessions, 4 attended 11, 2 came to 10 and 3 to 9. Figure 1CONSORT Flow Diagram. The baseline characteristics of the singing and control groups were well matched (Table 1). Four of each group were on long term oxygen therapy. In singers, there was a significant improvement in HAD anxiety score -1.1(2.7) vs. +0.8(1.7) p = 0.03 and SF-36 physical component score +7.5(14.6) vs. -3.8(8.4) p = 0.02. Breath hold time actually increased in the control group relative to the singing group -0.3(6.9) sec vs. +5.3(5.7) sec (p = 0.03). There was no significant difference in single breath counting, functional exercise capacity or recovery times following ISWT (Table 2). On direct questioning by the physiotherapist at the end of the study all participants in the singing group reported that they had found the workshops helpful, had practiced their singing at home and that they had found the singing helped them to control their breathing. Table 1 Baseline characteristics Total n = 28 Singing n = 15 Control n = 13 P value Age (yrs) 67.3 (8.1) 66.6 (9.3) 68.1 (6.8) 0.6 FEV 1 % predicted 37.2 (18.6) 36.8 (15.4) 37.7 (22.4) 0.9 Breath hold time (s) 26.3 (11.1) 27.4 (11.7) 24.9 (10.5) 0.9 Single breath counting (n) 23.4 (6.9) 24.8 (7.7) 21.8 (5.7) 0.3 HAD - anxiety score 5.8 (2.9) 6.3 (3.1) 5.3 (2.6) 0.4 HAD -depression score 5.8 (3.1) 5.7 (2.8) 5.8 (3.6) 1.0 SGRQ Total 48.4 (14.5) 51.1 (14.8) 45.3 (14.0) 0.3 SF36 PCS 36.8 (18.4) 32.0 (14.0) 42.2 (21.8) 0.1 SF36 MCS 52.0 (17.9) 47.4 (14.8) 57.2 (20.1) 0.2 ISWT (m) 198.6 (131.3) 174.0 (137.2) 226.9 (123.3) 0.3 Subjective recovery (s) 124.2 (77.6) 115.9 (50.4) 133.8 (102.0) 0.6 O 2 sat n recovery (s) 77.7 (71.6) 73.5 (44.6) 82.6 (95.7) 0.7 HR recovery (s) 131.0 (89.5) 97.6 (40.8) 169.6 (114.3) 0.03* FEV 1 - forced expiratory volume in one second, HAD - hospital anxiety and depression score, SGRQ - St George's Respiratory Questionnaire, SF36 short form 36, PCS - physical component score, MCS - mental component score, ISWT - incremental shuttle walk test distance, HR heart rate. *p < 0.05 unpaired t test singing group vs control group. Table 2 Changes from baseline Singing group n = 15 Control group n = 13 Δ breath hold time (s) - 0.3 (6.9) + 5.3 (5.7) 0.029* Δ single breath counting + 0.3 (7.7) + 2.0 (2.7) 0.46 Δ HAD anxiety - 1.1 (2.7) + 0.8 (1.7) 0.033* Δ HAD depression - 1.1 (2.5) - 0.1 (1.7) 0.21 Δ SGRQ Total - 1.1 (10.6) - 0.4 (5.6) 0.81 Δ ISWT (m) + 26 (52.6) + 11.3 (83.0) 0.58 Δ ISWT subjective recovery (s) + 9.9 (60.7) - 7.4 (81.7) 0.53 Δ O 2 Sat n recovery (s) + 47.3 (67.6) + 32.2 (124.7) 0.69 Δ HR recovery time (s) + 29 (63.8) + 19.4 (110.0) 0.78 Δ SF36 PCS + 7.5 (14.6) - 3.8 (8.4) 0.02* Δ SF36 MCS + 2.5 (20.9) - 3.2 (10.5) 0.38 HAD - hospital anxiety and depression score, SGRQ - St George's Respiratory Questionnaire, ISWT - incremental shuttle walk test distance, HR heart rate, SF36 short form 36, PCS - physical component score, MCS - mental component score. *p < 0.05 unpaired t test singing group vs control group. BODY.RESULTS.EVALUATION OF OPEN SINGING WORKSHOPS FOR RESPIRATORY PATIENTS: 150 patients attended the open workshops. 61% had never attended singing lessons or workshops previously. 96% rated the workshops as "very enjoyable" and 98% thought the workshop had taught them something about breathing in a different way. 81% of attendees felt a marked physical difference after the workshop. 92% of the participants said that they would like to attend a further singing workshop at the hospital. The remaining patients stated that the distance to the hospital would be the main reason for not attending. BODY.RESULTS.QUALITATIVE SURVEY OF PATIENT EXPERIENCE OF THE SINGING CLASSES: Eight patients who had participated in the singing groups were interviewed. The effect of the group was described by all in mainly positive terms and no participants reported any negative effects. Positive effects occurred in two main areas: physical and general well-being. Positive physical effects reported by the participants often related to the breathing training which was an integral part of the program, particularly "diaphragmatic breathing". This training brought a feeling of awareness and control which many of the participants found helped their breathing and eased symptoms of breathlessness. Singing training itself brought breathing control too; "It has made my life easier; I would have liked this when I was first diagnosed", "I increased my out breath from 4 to 14 counts", "I started breathing much better, from the stomach", "The exercises, thinking about breathing and relaxing when I have (breathing) problems....this has been very useful", "I always felt better afterwards physically". Other positive physical effects described were an impact on lifestyle and functional ability; "I have better posture now"; "Walking better, I go out more when it's not cold", "Now things are less of a chore, housework is no longer a struggle". Beneficial effects relating to general wellbeing also emerged. Relating to mood/pleasure participants reported that; "It was very enjoyable", "It opened up a new lease of life", "Emotionally......during singing it lifts you. I feel on top on the world. I also feel like that the day after. It makes COPD a lot easier to live with", "Its uplifting to sing...this diagnosis is gloomy so the psychological effect of the group is good". A feeling of community/social support was also reported; "Nice to have human contact, we achieved something together in the group", "I enjoyed the social contact it was great fun", "In six weeks we gelled as a group", "It felt good to be part of a team". Achievement/efficacy - many participants had carried aspects of the courses into daily life. Many of them continued to sing songs and perform the exercises from the group using printed sheets or a CD. It seems that learning to sing in a group brought a sense of achievement. "I do the exercises from the CD 2-3 times a week. I am looking for a local choir"; "I will keep it up; it's more enjoyable than other kinds of exercise"; "I learnt a lot, you are never too old to learn"; "The group taught me how and when to breathe; I got to relive my past (of singing) and get praise". Others reported that participating in the group had motivated them to reengage with pulmonary rehabilitation classes, or to take up exercises such as swimming and walking. One was having private singing lessons and several expressed a desire for such groups to be ongoing and made more available to patients with respiratory problems. BODY.DISCUSSION: The main findings of this study are that (1) the program of singing classes improved quality of life and anxiety but did not improve the control of breathing measures, or functional exercise capacity. (2) In interviews, patients who had participated in the trial reported benefits in their physical performance and general well-being as well as a sense of achievement and self-efficacy. (3) Individuals who chose to participate in the open singing sessions were overwhelmingly positive about the experience. BODY.DISCUSSION.SIGNIFICANCE OF FINDINGS: The idea that singing was beneficial for health goes back at least to the 19th century [24]. Two previous studies have looked specifically at singing in COPD patients. Bonilha et al randomised patients to either singing lessons, or a handicraft class once a week for 6 months [10]. Although singing practice produced an acute increase in inspiratory capacity, implying a reduction in gas trapping, it did not alter basal dyspnoea index (BDI). SGRQ improved equally in both groups and the singing group had an improvement in maximum expiratory pressure. Exercise capacity was not assessed. Engen studied 7 patients with emphysema who attended twice weekly vocal instruction classes for 6 weeks [23]. There was no change in spirometry, inspiratory muscle strength or exercise capacity, but patients had an improvement in single breath counting, which was associated with a change from a 'clavicular' to a 'diaphragmatic' pattern of breathing. In that study there was no control group and the teacher was not blind to the outcome measures. In the present study, anxiety score and physical component score of the SF36 improved and participants who were interviewed reported that they felt that the singing had been helpful in their everyday lives. HAD scores were not particularly high at baseline and patients were selected on the basis that they had symptomatic COPD and were able to attend, rather than because they were felt specifically to have a dysfunctional breathing pattern or psychological difficulties. It is possible that greater improvements might be found in particularly anxious sub-populations. The benefit in the physical rather than the mental component is consistent with the open workshop participants' reports that they felt physically different after the sessions. The immediate benefits reported by patients participating in the open workshops and from the interviews with singing patients are consistent with the previous observation that singing has an acute effect of reducing gas trapping [10]. Most reported sustained physical benefits, specifically identifying the value of learning breathing control. Several participants also mentioned that they felt that it was important to keep up the vocal exercises and singing in order to maintain any gains which may suggest that a longer period is necessary. The present study is also consistent with previous work that has found that singing training does not change, at least over the time courses explored to date, parameters such as exercise capacity [10,23]. We had expected that even if the singing class did not improve exercise tolerance it might hasten recovery, with patients adopting a more efficient breathing strategy. However, recovery time for heart rate, oxygen saturation or symptoms did not improve. Given the subjective benefits in physical sensation described by patients, the lack of change in exercise capacity is interesting. One possibility is that the improvements are entirely mediated in a psychological fashion. Since anxiety and depression are common and important co-morbidities in COPD [25,26] a novel approach to dealing with them could in any case be useful, particularly a non-drug treatment. An analogy would be the use of exercise prescriptions to treat depression [27]. Given the clear effects on wellbeing mentioned by the participants, one could also speculate that there might be benefits to efficacy and control in the absence of great physiological improvement. A patient's experience of their illness will be affected by a range of psychosocial factors in addition to their physical condition. All patients interviewed reported doing more singing and other pleasurable activities in their lives, suggesting that there may be long term benefits to participation in such a group. Consistent with this, singing has been used with some success in small studies to treat chronic pain where it seemed to improve coping [15] and after knee surgery [28]. A second possibility is that the 'dose' of singing was insufficient to alter conventional measures of breathing control or exercise capacity and that a longer or more intense period would be required to produce changes apparent in daily life. The singing teacher reported that she felt that after 6 weeks she was "only just starting to make progress" with many of the participants. It is interesting that in the current study, patients reported that what was identified to them as a 'diaphragmatic' breathing pattern was helpful. This involves facilitating outward movement of the abdomen while reducing upper ribcage movement during inspiration. It has been thought to be helpful in some patients, but although a reduction in oxygen cost of breathing has been noted [29], the approach has been associated with both an increased sensation of breathlessness and reduced mechanical efficiency in severe COPD [30,31] and is not recommended for routine use in the recent British Thoracic Society/Association for Chartered Physiotherapist in Respiratory Care(BTS/ACPRC) guidelines on "Physiotherapy management of the adult, medical, spontaneously breathing patient" [12]. The improvement in breath hold time observed in the control group was unexpected. A strength of the study was that the singing teacher was blind to the outcome measures used so could not "teach to the test". The teacher speculated that the singing group may have learnt to take a more controlled or comfortable breath and therefore smaller breath in, leading paradoxically to a reduction in breath hold time. Hyperventilation has been shown to increase breath hold time in patients with respiratory disease which might also be relevant [22]. If the benefits of participating in a singing group are largely psychological, it would suggest that attention would need to be focused on the aspects of the singing that addressed this, or even on the choice of material, rather than focussing on particular nuances of technique. Singing is, of course, likely to be a therapy that suits some people and not others, and the benefits that accrue are likely to be greatest in those who enjoy the experience. This is to some extent different from pulmonary rehabilitation where the health benefits of exercise are generally accepted. As with pulmonary rehabilitation the physiological and psychological effects could be complementary. A limitation of our study was the absence of an active control group. The previous paper of Bonilha et al. showed similar improvements of quality of life in patients submitted to singing training or handcraft artwork [10], so the observed improvements in anxiety and quality of life in the trial may have been due to regular contact with a social group rather than the singing specifically. However, in our study patients reported immediate positive effects on well being (probably because of a reduction in dynamic hyperinflation [10]) and this may therefore have reinforced participation in singing groups (as opposed to other 'social activities' which, although social have no physical effect), making the provision of singing classes a good strategy for reducing social isolation, a significant problem in chronic respiratory disease [32]. BODY.CONCLUSIONS: Singing lessons improved anxiety and the physical component score of the SF36 but did not improve measures of breathing control, functional exercise capacity or recovery time. Participants reported that they found the singing beneficial and reported positive changes in their physical ability and wellbeing. It is likely that the effects of singing training will vary between individuals, but that it will be a positive experience for those who choose to take part. Further work is needed to quantify the magnitude and duration of improvement benefits, to set them in context against the resource implications of making singing groups for patients more widely available as a palliative therapy. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS' CONTRIBUTIONS: NSH, VML, VJH, PC and MIP conceived the study; VML, PC, EJF, AE and JLK performed the study measurements. VML, VJH, AE and NSH performed the data analysis. VML wrote the first draft of the paper to which all authors subsequently made contributions. All authors read and approved the final manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2466/10/41/prepub BODY.SUPPLEMENTARY MATERIAL: Additional file 1Help Yourself - physiotherapy for people with respiratory symptoms". Standard booklet given to all patients participating in the trial.Click here for file Additional file 2Singing for breathing - description of program. Detailed description of singing program, selection of materials and conduct of classes.Click here for file Additional file 3Open sessions satisfaction survey. Survey used for participants to assess the open singing sessions.Click here for file Additional file 4Structured interview template. Template used to guide assessments by psychologists following the singing trial.Click here for file

TITLE: Effect of a randomised exclusive breastfeeding counselling intervention nested into the MINIMat prenatal nutrition trial in Bangladesh ABSTRACT.ABSTRACT.AIM: It is unknown whether maternal malnutrition reduces the effect of counselling on exclusive breastfeeding. This study evaluated the effect of breastfeeding counselling on the duration of exclusive breastfeeding, and whether the timing of prenatal food and different micronutrient supplements further prolonged this duration. ABSTRACT.ABSTRACT.METHODS: Pregnant women in Matlab, Bangladesh, were randomised to receive daily food supplements of 600 kcal at nine weeks of gestation or at the standard 20 weeks. They also were allocated to either 30 mg of iron and 400 μg folic acid, or the standard programme 60 mg of iron and folic acid or multiple micronutrients. At 30 weeks of gestation, 3188 women were randomised to receive either eight breastfeeding counselling sessions or the usual health messages. ABSTRACT.ABSTRACT.RESULTS: The median duration of exclusive breastfeeding was 135 days in the counselling group and 75 days in the usual health message group (p < 0.001). Prenatal supplements did not modify the effects of counselling. Women in the usual health message group who were randomised to multiple micronutrients exclusively breastfed for 12 days longer than mothers receiving the standard iron–folate combination (p = 0.003). ABSTRACT.ABSTRACT.CONCLUSION: Breastfeeding counselling increased the duration of exclusive breastfeeding by 60 days. This duration was not influenced by the supplements. BODY: Key notes Earlier studies of breastfeeding counselling have shown positive effects on duration of exclusive breastfeeding, but it is not known whether prenatal nutrition interventions increase this effectEight counselling sessions increased exclusive breastfeeding by 60 days, but timing of prenatal food supplementation and different micronutrient alternatives did not increase the durationAmong women who did not receive the breastfeeding counselling, prenatal multiple micronutrient supplementation was associated with 12 days longer exclusive breastfeeding that may be of small public health importance BODY.INTRODUCTION: Exclusive breastfeeding for the first six months of life, and continued breastfeeding together with adequate and safe complementary feeding for two years, is critical for physical well‐being, cognitive development and child survival. Exclusive breastfeeding until six months of age could reportedly prevent more than 800 000 child deaths worldwide every year 1. In Bangladesh, around 90% of mothers are still breastfeeding their babies at one year of age and 88% are still breastfeeding at around two years 2. Despite breastfeeding promotional activities over the last two decades, the prevalence of exclusive breastfeeding at four to five months of age has remained relatively small, at 32% 2. Rapid urbanisation and female employment could partly explain the introduction of other foods before the age of six months. Many women in rural areas, and those who are not employed, introduce milk or semisolid foods to children at less than three months of age. Earlier studies reported that a high proportion of the mothers felt that they did not have enough breastmilk 3. Peers and other family members, such as mothers‐in‐law or elderly family members, could also influence perceptions of insufficient breastmilk. Studies in Bangladesh have documented a significant increase in exclusive breastfeeding as a result of community‐based peer counselling 4 or breastfeeding counselling in hospitals 5. A systematic review and meta‐analysis confirmed that peer counselling enhanced the duration of exclusive breastfeeding, although the contextual influence of a formula‐feeding tradition might have decreased the effect 6. A wide range of counselling and breastfeeding promotion activities have been shown to increase the duration of exclusive breastfeeding 7. Continuous, repeated interactive sessions with counsellors in the health system were reported to be effective by a systematic review 8. Despite this, our knowledge is limited regarding the optimal timing, dose and mode of counselling that could be scaled‐up as an integrated part of the continuum of care provided by the health system to mothers and their children 8. Furthermore, it has been questioned whether underweight mothers are less able to breastfeed their infants successfully 9. A review of multiple micronutrient supplements provided to breastfeeding women was inconclusive regarding the effects on breastfeeding performance due to the lack of good‐quality studies 10. To the best of our knowledge, there have been no previous studies that have evaluated the effect of prenatal food and multiple micronutrient supplementations on later breastfeeding performance. The Maternal and Infant Nutrition Interventions (MINIMat) was a randomised trial in Matlab in rural Bangladesh (International Standard Randomised Control Trial Number: ISRCTN16581394). It was designed to examine the effect of combined food and micronutrient interventions on haemoglobin, birthweight and infant survival 11. An exclusive breastfeeding counselling intervention was nested into the trial and was provided to consenting mothers at around week 30 of pregnancy. We postulated that in a population where being underweight was common, the early timing of prenatal food supplements combined with multiple micronutrients would enhance the effect of exclusive breastfeeding counselling. The aim was to assess the effect of eight breastfeeding counselling sessions from late pregnancy to early infancy on the prevalence and duration of exclusive breastfeeding and to evaluate whether this effect was further increased by an early invitation to prenatal food supplements versus the usual timing and supplements with multiple micronutrients instead of the standard iron and folate combination. BODY.METHODS: The MINIMat trial was conducted in Matlab, a rural subdistrict 57 km south‐east of Dhaka, the capital of Bangladesh. The International Centre for Diarrhoeal Diseases Research, Bangladesh (icddr,b), maintains the Health and Demographic Surveillance System in Matlab that monitors demographic and selected health information in the area. A total of 4436 pregnant women were enrolled from November 2001 to October 2003 and were randomised to the different nutritional interventions. The infants born to these women have been followed since birth. Written, informed consent was obtained from all of the participating mothers, and the study was approved by the Research and Ethical Review Committees of icddr,b. BODY.METHODS.STUDY DESIGN: The MINIMat trial had a factorial design with two food supplement alternatives and three micronutrient supplements. At around week 30 of pregnancy, consenting women were allocated to breastfeeding counselling or received the usual antenatal healthcare messages. This study focuses on the effect of these interventions on the prevalence and duration of exclusive breastfeeding. BODY.METHODS.RANDOMISATION: In the MINIMat trial, all pregnant women were individually randomised to one of the food supplement groups and to one of the three micronutrient interventions, as previously described 11. The following eligibility criteria had to be met for enrolment: viable foetus, gestational age of less than 14 weeks by ultrasound examination, no severe illness and written consent for participation. At around nine weeks of pregnancy, women were randomly invited to receive food supplements at around nine weeks of gestation or to the standard programme where they could start the supplements when they chose, usually at around 20 weeks of gestation. Food supplements of 600 kcal per day was made available through community nutrition centres six days per week and continued up to the end of pregnancy. At 13 weeks of gestation, women started to receive the randomly allocated micronutrient supplementations that continued for their entire pregnancy: (i) 30 mg Fe fumarate and 400 μg folate, (ii) the standard 60 mg Fe fumarate and 400 μg folate or (iii) multiple micronutrients with 15 micronutrients including 30 mg Fe fumarate and 400 μg folate. At around 30 weeks of gestation, simple random sampling was used to allocate consenting pregnant women to receive either counselling on exclusive breastfeeding or to receive the usual health message delivered by the regular icddr,b health staff during the antenatal clinic visits. The micronutrient supplementation was double‐blinded, while the food supplementation and the later breastfeeding counselling were randomly allocated but not blinded. An independent statistician performed all the randomisation procedures using a computer program. Randomisation lists were generated that included participant identity codes of the Health and Demographic Surveillance System and codes for the allocated interventions. Field staff, supervised by study supervisors, carried out the practical allocation to the different interventions. There were no deviations from the randomly decided allocations. Randomisation codes were safely kept at the administrative office of icddr,b and were not accessed until after performing the intent‐to‐treat analyses of the primary outcomes. BODY.METHODS.EXCLUSIVE BREASTFEEDING INTERVENTION: Nine women were recruited and trained as breastfeeding counsellors. Five were recruited at the start of the study, and another four joined after six months. They all lived in the local community, the Matlab study area, were married with at least one child, had breastfeeding experience and had a bachelor's degree equivalent to 14 years of schooling. One of the authors (IK) and one senior breastfeeding counsellor trained the other counsellors using a 40‐hour World Health Organization (WHO) and United Nations Children's Fund Breastfeeding Counselling Training module, which had been translated into the local Bangla language and adjusted to the local context. This training module had been successfully used in previous studies 4. The training was provided in daily four‐hour sessions over ten days. Counselling skills were taught using demonstrations and role‐play and included listening to mothers, learning about their difficulties, assessing the position and attachment of babies during breastfeeding, building mothers' confidence, giving support and providing relevant information and practical help when required. The training also included practical sessions with pregnant women and mothers with infants. Two senior breastfeeding counsellors with at least six years of experience in counselling and one of the authors (IK) supervised the fieldwork and provided practical help at fortnightly sessions or via cell phones when required. All women received basic healthcare messages, as is the usual practice in this region of Bangladesh. The women allocated to breastfeeding counselling received eight sessions: two sessions during the last trimester of pregnancy, one session in the seven days after delivery and five sessions at monthly intervals up to six months after childbirth. Counsellors were free to make additional contact if required. Counselling was given individually at home, but key family members were also included. The duration of each counselling visit was typically 20–40 minutes, depending upon the mother's lactation stage and her individual needs. Usually, the first two to three visits were longer, to build the mothers' confidence, and the later counselling visits were shorter as they aimed to just give support to mothers and reinforce the health messages. The women in the usual health message group received the messages on breastfeeding practices that were provided by the health staff of icddr,b when pregnant mothers visited the subcentres for their regular antenatal visits. These messages included the benefits of colostrum, advice on exclusive breastfeeding for four to six months and the recommended starting of complementary feeding from four to six months along with continuing breastfeeding for two years. BODY.METHODS.OUTCOME DATA: Trained female interviewers collected data on infant feeding status every month up to 12 months of age. Data collection interviews were carried out one to three days before the counselling visits. A precoded semistructured feeding chart was developed, and feeding during the last 24 hours was recorded. Furthermore, the mother was asked whether the baby had received anything else besides breastmilk during the last month. If the mother had given the infant any other food or liquid than breastmilk for two consecutive days or more during the last month, this information was considered when classifying the feeding status and coded as partially breastfed. BODY.METHODS.FEEDING CLASSIFICATION: The WHO definitions were used. To be classified as exclusive breastfeeding during the last month, only breastmilk could be given, not even water. Oral rehydration solution, liquid medicines and vitamins were allowed within the definition of exclusive breastfeeding. BODY.METHODS.OTHER DATA: Maternal nutritional status and information regarding place and mode of delivery were extracted from the MINIMat databases 11. The socio‐economic characteristics of the households were determined using a continuous household asset score previously generated for this population, including data on land ownership, the construction materials used for house walls and the ownership of household assets. Data on the presence of such characteristics were analysed by principal component analysis, and a score was generated for each household, according to the principles that have been used by the World Bank and several other organisations 12. BODY.METHODS.DATA QUALITY AND MANAGEMENT: An additional team cross‐checked one‐tenth of the interviewers' scheduled visits to ensure the quality of the collected data. The questionnaires were checked daily, and, if the information was incomplete or not clear, a supervisor returned to the home the next day to complete and clarify the information. Data were stored in relational databases and further checked for any inconsistencies that were corrected in comprehensive data cleaning procedures. BODY.METHODS.STATISTICAL METHODS: The chi‐square test was used to compare the proportions receiving colostrum and the effect of the randomised breastfeeding intervention, namely the breastfeeding counselling versus the usual health message, on exclusive breastfeeding prevalence at four and six months. The total duration of exclusive breastfeeding was assessed in an intent‐to‐treat analysis, by including every participant in the analysis according to their initial randomisation. The analyses of the duration of exclusive breastfeeding were performed by analysis of variance (ANOVA). Interactions between prenatal food and micronutrient groups and the breastfeeding counselling intervention were analysed using product terms in ANOVA. First, a three‐way interaction was examined as follows: exclusive breastfeeding counselling × food supplementation × micronutrient supplements. If this was not statistically significant, two‐way interactions were examined as follows: exclusive breastfeeding counselling × food supplementation and exclusive breastfeeding counselling × micronutrient supplements. The significance level for interactions was set at p = 0.10. BODY.METHODS.SAMPLE SIZE: The sample size for the MINIMat trial had been calculated for one of the primary outcomes, that is the effect of food and micronutrient supplements on birthweight. The sample size available for this study provided a power of 0.90 (type II error) and a probability of 0.05 (type I error) for a 6% difference in the prevalence of exclusive breastfeeding for the counselling group compared to the usual health message group. Such a difference was much smaller than those observed in previous studies. The sample size available for this study also provided a power of 0.88 (type II error) and a probability of 0.05 (type I error) for a 10% difference in the prevalence of exclusive breastfeeding for simple effect comparisons between any two of the six groups defined by the combination of the two counselling groups and the three micronutrient groups. BODY.RESULTS: A total of 3188 mothers were randomised either to receive exclusive breastfeeding counselling from the counsellors or to receive the usual health messages from the Matlab regular health staff. The latter group served as the control. After omissions, 1387 mother–infant pairs in the counselling group and 1402 mother–infant pairs in the usual health message group completed six months of follow‐up. The distribution of pregnant women and their children in the exclusive breastfeeding counselling groups and the numbers lost to follow‐up are shown in Figure 1. The main reason for the women's lack of availability at the time of randomisation, at week 30, was the custom for pregnant women to visit their parents' home at this time. Figure 1Study flow chart. The distribution of pregnant women and their children in the exclusive breastfeeding (EBF) counselling groups and the numbers lost to follow‐up during the Maternal and Infant Nutrition Interventions in Matlab (MINIMat) trial. Mothers in the randomisation groups were similar at baseline (Table 1). The average age of the mothers was 26 years, their mean body weight was 45 kg, and their average height was 150 cm. Socio‐economic variables were similar across groups. The mean birthweights were 2690 and 2697 g and the birth lengths were 47.7 and 47.6 cm in the breastfeeding counselling and usual health message groups, respectively. About 57% and 56% had been delivered at home. The mean and standard deviation (SD) gestational age at birth was 38.7 ± 1.8 weeks and 38.7 ± 1.7 weeks in the breastfeeding counselling and usual health message groups, respectively. Table 1 Baseline characteristics of mothers and infants at birth in the two intervention groups Characteristic Breastfeeding counselling group (n = 1387) Usual health message group (n = 1402) Maternal Age (years) 25.8 ± 6.0 25.7 ± 5.8 Education (mean number of years completed in formal education) 7.1 ± 2.9 7.2 ± 2.9 Asset score −0.10 ± 2.30 0.06 ± 2.33 Asset score (n/n % in lowest quintile) 291/1417 (20.5) 278/1428 (19.5) Asset score (n/n % in highest quintile) 260/1417 (18.3) 303/1428 (21.2) Height (cm) 149.7 ± 5.3 149.8 ± 5.4 Weight at 8 weeks of gestation (kg) 45.1 ± 6.8 45.4 ± 6.9 BMI (at 8 weeks of gestation) 20.1 ± 2.7 20.2 ± 2.7 Place of delivery (n/n % at home) 808/1417 (57.0) 795/1428 (55.7) Type of delivery (n/n % Caesarean section) 63/1417 (4.4) 59/1428 (4.1) Infant Sex (n/n % female) 684/1417 (48.3) 699/1428 (48.9) Birthweight (g) 2690 ± 415 2697 ± 409 Birth length (cm) 47.7 ± 2.2 47.7 ± 2.1 Data are mean (SD) unless indicated to be n/n (%). John Wiley & Sons, Ltd In the breastfeeding counselling group, 95.5% of the babies had received colostrum as the first food, compared with 85.8% in the usual health message group (p < 0.001). By 72 hours, almost all babies in both groups had been breastfed. The median duration of exclusive breastfeeding was longer in the breastfeeding counselling group (135 days, 95% CI: 131–139) than in usual health message group (75 days, 95% CI: 68–82) – a difference of 60 days (p < 0.001). At four months, 69.0% (95% CI: 66.1–71.9) of the breastfeeding counselling group were being exclusive breastfed, as were 46.6% (95% CI: 42.8–50.4) in the usual health message group, while the corresponding figures at six months were 15.3% (95% CI: 10.4–20.1) and 6.4% (95% CI: 1.3–11.5), respectively. There was no statistically significant three‐way interaction between breastfeeding counselling, food supplements and micronutrient supplements. The timing of the food supplements did not influence the effect by counselling on the duration of exclusive breastfeeding, with a p for interaction of 0.115 (Table 2). Table 2 Analysis of breastfeeding counselling intervention with food and micronutrient supplementations on duration of exclusive breastfeeding Interventions n Mean duration 95% CI p value for interaction Analysis of breastfeeding counselling × food supplementation 0.115 BFC + Early food 699 108.8 104.3–113.3 BFC+ Usual food 688 113.7 109.2–118.1 UHM+ Early food 708 77.3 72.8–81.8 UHM+ Usual food 694 75.0 70.5–79.5 Analysis of breastfeeding counselling × micronutrient supplementation 0.064 BFC+ Fe30 451 111.4 105.8–116.9 BFC+ MMS 447 111.2 105.6–116.8 BFC+ Fe60 489 111.1 105.8–116.4 UHM+ Fe30 466 73.3 68.0–78.6 UHM+ MMS 467 83.7 78.1–89.2 UHM+ Fe60 469 71.6 66.0–77.1 Interventions: BFC = breastfeeding counselling; UHM = usual health message; Early food = early invitation to food supplementation (around week 9), Usual food = usual timing of start of food supplementation (around week 20), Fe30 = 30 mg Fe and folic acid, Fe60 = 60 mg Fe and folic acid, MMS = multiple micronutrients. John Wiley & Sons, Ltd Multiple micronutrient supplements did not further increase the effect of counselling on the duration of exclusive breastfeeding (Table 2). In the group of mothers who did not receive counselling, multiple micronutrient supplements were associated with longer exclusive breastfeeding of 12 days (p = 0.003). BODY.DISCUSSION: We have shown that one‐to‐one breastfeeding counselling with eight sessions from pregnancy to six months after delivery with trained local counsellors resulted in higher prevalence and longer duration of two months of exclusive breastfeeding. The comparison group received the usual health messages that also included some breastfeeding promotion. Data were longitudinally collected by interview at home every month, reducing the risk of recall bias. We also conducted a validation study using the deuterium dilution technique of mothers' reported feeding behaviour and found a strong association between the questionnaire‐based information and the validation data, with positive predicted value of 0.84 13. As shown in reviews of breastfeeding counselling interventions, continued counselling, support over time and contextual factors influence the magnitude of effects 6, 7, 8. In a previous peer‐counselling trial in urban Dhaka, the dose of the intervention, namely the number of counselling sessions, was almost double the number in the present study, and the size of the effect was larger 4. The present study showed that a lower number of contacts, close to the number of contacts a pregnant and delivering woman usually has with the regular maternal and infant health services, also resulted in a substantial increase in the duration of exclusive breastfeeding. The optimal number of counselling sessions across late pregnancy and early infancy is not known 6. Importantly, the training provided in this study emphasised the interactive character of the sessions 8 and the continuity of contact between the counsellor and the mother. The total duration of exclusive breastfeeding in this trial may also have been influenced by the previous WHO guideline of exclusive breastfeeding four to six months that was still Bangladesh government policy at the time of the study. Previous studies have asked whether marginally nourished women have the needed lactational capacity to breastfeed. A study in Bangladesh found evidence that the volume and quality of breastmilk in marginally nourished women may still be sufficient to ensure the healthy growth of their infants 9. A Cochrane review departed from the fact that many lactating women in low‐income settings suffer from multiple micronutrient deficiencies that might impair lactation performance. The authors found no trials, and only a few other studies, that addressed the possible impact of multiple micronutrients on breastfeeding outcomes and the result was nonconclusive 10. In this study, we hypothesised that prenatal multiple micronutrient supplementation versus iron–folate along with breastfeeding counselling would further increase the duration and rates of exclusive breastfeeding and providing prenatal food supplements earlier would further influence the effect in a positive way. In the group randomised to receive breastfeeding counselling, neither the multiple micronutrients nor the timing of the prenatal food supplementation affected exclusive breastfeeding duration. In the control group that was randomised to receive the usual health message, the allocation of multiple micronutrients was associated with longer duration of 12 days of exclusive breastfeeding. Even if this is valid, the public health importance of such a small difference might be limited. BODY.CONCLUSION: To conclude, the relatively limited number of eight counselling sessions with trained counsellors from pregnancy to the first part of infancy increased exclusive breastfeeding by two months. This is a 'dose' of the intervention that could be part of regular antenatal and child health services in order to achieve better adherence to the recommended duration of exclusive breastfeeding 1. However, the timing of a prenatal food supplementation and different micronutrient alternatives did not increase the effect of counselling. Among women who did not receive breastfeeding counselling, multiple micronutrients were associated with slightly longer exclusive breastfeeding that most likely may be of a small public health importance. BODY.CONFLICTS OF INTEREST: The authors declare that they have no competing interests. BODY.FUNDING: The MINIMat research study was funded by icddr,b, the United Nations Children's Fund, the Swedish International Development Cooperation Agency, the UK Medical Research Council, the Swedish Research Council, the Department for International Development, the Japan Society for the Promotion of Science, the Child Health and Nutrition Research Initiative, the Uppsala University and United States Agency for International Development. The funders had no role in any aspect of the study design or resulting manuscript.

TITLE: Social Presence and Use of Internet-Delivered Interventions: A Multi-Method Approach ABSTRACT.OBJECTIVE: Internet-delivered interventions can effectively change health risk behaviors and their determinants, but adherence to intervention websites once they are accessed is very low. This study tests whether and how social presence elements can increase website use. ABSTRACT.METHODS: A website about Hepatitis A, B, and C virus infections was used in a preparatory lab-based eye-tracking study assessing whether social presence elements attract participants' attention, because this is a prerequisite for affecting website use. In the following field study, 482 participants representative of the Dutch population were randomized to either a website with or a website without social presence elements. Participants completed a questionnaire of validated measures regarding user perceptions immediately after exposure to the website. Server registrations were used to assess website use. ABSTRACT.RESULTS: Participants in the experimental condition focused on the social presence elements, both in terms of frequency (F(1, 98) = 40.34, p<.001) and duration (F(1, 88) = 39.99, p<.001), but did not differ in website use in comparison with the control condition; neither in terms of the number of pages visited (t(456) = 1.44, p = .15), nor in terms of time on the website (t(456) = 0.01, p = .99). ABSTRACT.CONCLUSIONS: Adding social presence elements did not affect actual use of an intervention website within a public health context. Possible reasons are limited attention for these elements in comparison with the main text and the utilitarian value of intervention websites. BODY.INTRODUCTION: Internet-delivered interventions can effectively change health risk behaviors and their determinants [1], but the actual use of these interventions by the target group once they access the website is very low [2]. Eysenbach [3] even defined the "law of attrition", which considers high attrition rates as a natural and typical feature of Internet-delivered interventions. For example, server statistics of an intervention promoting heart-healthy behaviors showed that 285,146 visitors from unique IP addresses accessed the home page in a 36-month period, but 56.3% of them left the intervention website within 30 seconds [4]. This finding touches upon a critical issue in Internet-delivered interventions: how can they ever have a public health impact if people only briefly use the actual intervention? It is therefore relevant and necessary to focus on factors related to the use of an Internet-delivered intervention once people arrive at the intervention website (i.e., website use) [5]. These factors relate to the visitor (e.g., people's motivation to be healthy [6], [7]) as well as the intervention website (e.g., offering tailored information [8], [9]). Not only is the content of the website important [10], but also the specific characteristics of the website itself. The current study aims to systematically manipulate a website characteristic – i.e., social presence – and link this to (1) website use and (2) user perceptions as the working mechanisms behind this possible effect of social presence. The rationale behind this aim is explained in the follow paragraphs, resulting in hypotheses to be tested in the current study. The focus of the current study is on social presence, which can be seen as the extent to which a medium is perceived to convey a feeling of human contact, sociability, and sensitivity [11]. This is realized by adding social presence elements: presenting human images [12] and testimonials [13] in which people share their experiences and ideas regarding the information presented on the website with others. Previous studies revealed that adding these social presence elements increases perceived social presence and positively affects attitude towards shopping websites [14] as well as intention to use them [13]. Two issues remain unclear, however. First, whether social presence also has added value in a public health context. Second, whether the positive effect regarding attitude and intention to use also hold when looking at the actual behavior (i.e., website use) instead of its precursors. Given that intention is the most important predictor of actual behavior [15], this is hypothesized to be the case. Thus, social presence is expected to increase website use in a public health context (Hypothesis 1). What are the working mechanisms behind this possible effect of social presence on website use? The main idea is that social presence results in a positive user experience, which increases website use. User experience refers to what a person thinks and feels during and after being exposed to a website [16] and is deemed even more important than usability, which only concerns ease of use [17]. User experience consists of both cognitive perceptions (e.g., efficiency, effectiveness) and affective perceptions (e.g., enjoyment) [18]. Efficiency refers to easy search and access of information provided and effectiveness refers to the quality of that information (e.g., in terms of relevance) [19]. These cognitive perceptions have parallels with perceived ease of use and perceived usefulness in the technology acceptance model, but are applicable in a broader context [20]. The affective perceptions of user experience are often referred to as enjoyment [21], [22]. Active trust refers to the confidence in acting on the provided information on a website, which can result in increased website use [23]. Efficiency, effectiveness and enjoyment can be expected to be positively associated with website use and these associations are hypothesized to be partly mediated by active trust, which is in line with the limited evidence that is currently available [24], [25] (Hypothesis 2). Previous studies investigating social presence and user perceptions consistently demonstrated that social presence positively affects perceptions of both effectiveness and enjoyment regarding shopping websites [13], [14], [26]. The current study extends previous studies by including a measure of interest besides enjoyment, because this is a motivational factor that "guarantees the person's engagement" [27]. Although interest is sometimes defined as feelings of enjoyment [28], interest and enjoyment differ in critical ways [29]. Experiments using several kinds of stimuli (e.g., random polygons, poetry, and experimental paintings) indicate that enjoyment is not a necessary condition for interest [30]. The two antecedents of interest are, according to Silvia [29], appraisal of coping potential (e.g., that people can deal with the presented information due to the use of everyday language) and appraisal of novelty-complexity (e.g., due to the use of imagery). The previously discussed social presence elements are in line with Silvia's [29] suggestion to use imagery (e.g., human images) and everyday language (e.g., testimonials) to positively affect appraisal of novelty-complexity and coping potential respectively, resulting in increased interest. Moreover, increased interest results in spending more time reading information on a topic [31]. An initial hypothesis would therefore be that interest due to adding social presence elements increases website use (Hypothesis 3a). However, the increased complexity due to adding social presence elements can also have a negative impact. Previous studies demonstrated that complexity has a negative effect on attitude towards a website and therewith on intention to use [32], [33], pleading for simplicity of websites [34]. An alternative hypothesis would therefore be that adding social presence elements decreases website use because of increased complexity (Hypothesis 3b). BODY.INTRODUCTION.WEBSITE: A website about Hepatitis A, B, and C virus (HAV, HBV, HCV) infections was used to test the effect of adding social presence elements on website use and the working mechanisms behind this possible effect. These infections all affect the liver, but there are differences in terms of mode of transmission, consequences, and prevention. Besides the home page, the website consisted of four pages per Hepatitis virus. The first page introduced the virus briefly and the other three pages outlined transmission, consequences, and prevention respectively. This resulted in a total of twelve pages of website content (for all virus types) besides the home page. The content for these pages was based on information from the Dutch National Hepatitis Centre. The content was text-based, purely informative, non-tailored, and very brief (i.e., 5–10 lines of text per page). Use of this website has been proven to result in increasing practical knowledge regarding Hepatitis [25], which is currently very low in the Netherlands [35]. The experimental website for this study was created by adding social presence elements to the website previously used, which served as the control website in this study. BODY.INTRODUCTION.SOCIAL PRESENCE ELEMENTS: Social presence elements were based on previous studies and consisted of adding human images (of people varying in age and gender) [12] and testimonials [13] in which people shared their experiences and ideas regarding the information presented on the website with the visitor. The above social presence elements were added to the home page and three pages per virus type that outlined transmission, consequences, and prevention respectively. Therefore, a total number of ten pages contained social presence elements. The number of pages of both website versions were identical. Previous research proved that JPEG file sizes are a good proxy for complexity of web pages [36]. File sizes are not only related to complexity, but also to page size (i.e., resolution). Therefore, screenshots of pages to which social presence elements were added in the experimental website, were captured for both websites at the same resolution (1024×768 pixels). The screenshots of the experimental website were – according to the JPEG file sizes – more complex in comparison with the control website (218 vs 137 kB, t(18) = 22.42, p<.001), which is a presumption in Hypothesis 3b (i.e., adding social presence elements increases complexity). BODY.INTRODUCTION.PREPARATORY STUDY: The aim of the preparatory study was to assess whether social presence elements attract participants' attention, because this is a prerequisite for them to affect user perceptions and (intention to) use. A previous study – based on self-reports – found a positive relationship between attention and user perceptions [37]. Eye-tracking, however, is a more suitable method to assess the course of attention over time [38]. This preparatory study built upon previous studies that successfully applied eye-tracking with regard to perceptions of health-related information [39] and websites [12]. There is general agreement on the strong association between eye movements and attention [40]. The idea behind eye-tracking is that saccadic eye movements are associated with shifts in attention. Fixations refer to the periods between saccadic eye movements in which gaze is held almost stationary. BODY.METHODS.PARTICIPANTS: Participants were a convenience sample of undergraduate students that received course credits for their participation in this preparatory study. Participants were screened to ensure normal or corrected vision, and those wearing hard contact lenses or eyeglasses were excluded from participation, because this can result in difficulties in capturing eye movements. A total of 24 participants were randomized (16 women, 8 men). Mean age of the sample was 20.1 years (SD = 1.8). Participants were recruited through a research panel of a Dutch Internet research agency; therefore, they could be considered computer literate. From this panel, a stratified sample of 982 potential participants was invited to participate in a study about Hepatitis through email. Informed consent was obtained online, which is the regular procedure for this research panel. This sample was representative of the Dutch Internet population above 18 years, taking into account gender, age, and level of education. BODY.METHODS.DESIGN AND PROCEDURE: After explaining the study procedure and signing informed consent, participants were seated approximately 25 inches from a computer screen. The laboratory room was artificially lit and there was no influx of sunlight. First, the eye-tracker system (more detailed information in the Apparatus section) was calibrated. Subsequently, participants were told that we were interested in their opinion to improve a website and that they could freely explore this website. Participants were randomly assigned to two conditions. The control condition consisted of the existing website as used in a previous study [25]. The experimental condition added social presence elements to this website (as described above). A brief questionnaire had to be completed immediately following exposure to the website and consisted of measures regarding interest and perceived social presence as a manipulation check. It was stressed that there were no right or wrong answers. Participants were randomly assigned to either the control condition or the experimental condition (Figure 4). Both conditions were similar to those in the preparatory study. The pre-test (i.e., before being exposed to the website) consisted of a Hepatitis knowledge questionnaire. After the pre-test, participants were directed to their assigned version of the website. Participants were asked to base their opinion about the website on their first impression and were told they could freely explore the website until they started completing the post-test (immediately after visiting the website). The objective was not to force participants to thoroughly study the website, but to mimic a real-life situation in which time and opportunity to invest in the website is limited [4]. The post-test consisted of measures regarding user perceptions and perceived social presence as a manipulation check. For these measures, it was stressed that there are no right or wrong answers. One week later, participants were invited to complete the follow-up measure, which was the same as the pre-test (i.e., the Hepatitis knowledge questionnaire). The follow-up measure was added to test whether participants did not only click through the website but actually processed and memorized its content (i.e., by looking at both within- and between-group differences regarding Hepatitis knowledge over time). 10.1371/journal.pone.0057067.g004Figure 4Flowchart of study design and attrition. Participants received an incentive (i.e., credit points for panel members) to participate in the study, which represented a value of € 2.50. Panel members can save credit points over time, which can be exchanged for online vouchers valid in several stores in the Netherlands. Relevant ethical safeguards were met with regard to the participant confidentiality and consent. BODY.METHODS.ETHICS STATEMENT: The Simon Fraser University Research Ethics Board granted ethical approval (file number: 2012s0168). BODY.METHODS.APPARATUS: The eye-tracker system used was Applied Science Laboratories Model 504 with head tracking integration. Eye movements were processed using an autofocus eye camera mounted on a optics mechanism (max. 100° pan, 25° tilt), which was positioned under the computer screen. The camera has eye illumination that consists of a ring of near-infrared light-emitting diodes. The sampling rate was 60 Hz and the measurement error was <1.0°. Participants wore a headband with a mounted sensor, which allowed tracking of head movements without loss of eye image. This permitted participants to move their heads in a relatively natural manner. Gaze Tracker software was used to capture and process eye-tracking data (measures are explained in the following section). BODY.METHODS.MEASUREMENTS: The following measurements were used within the preparatory study. The following measurements were used within the main study. BODY.METHODS.MEASUREMENTS.ATTENTION: Attention is assessed by the number of fixations and total fixation duration in milliseconds (ms) within each area of interest on the website as defined by rectangular regions called lookzones. There were two lookzones on each page; the main text and the social presence elements (Figure 1). The main text lookzone was similar for both conditions. The threshold for minimal duration for a fixation was set at 50 ms [40]. 10.1371/journal.pone.0057067.g001Figure 1Example of lookzones on a page (image has been blurred to ensure anonymity). BODY.METHODS.MEASUREMENTS.INTEREST: In line with Turner and Silvia [30], two items measured interest (uninteresting-interesting and boring-engaging; alpha = .72) on a 7-point Likert scale. BODY.METHODS.MEASUREMENTS.PERCEIVED SOCIAL PRESENCE: Five items (e.g., "There is a sense of human warmth in the website"; alpha = .87) were used to measure perceived social presence as a manipulation check [12], [41]. Items were answered on a 7-point Likert scale ranging from (1) 'strongly disagree' to (7) 'strongly agree'. Similar to the preparatory study, five items (alpha = .95) were used to measure perceived social presence as a manipulation check. BODY.METHODS.ANALYSES: Data from participants who encountered technical problems (e.g., difficulties in capturing eye movements) or artifacts (e.g., too many blinks) resulting in >25% missing eye-tracking data were excluded from the analyses regarding attention (n = 5). Mixed models were used to test for differences in the measures of attention, using page as a within-subject factor and condition as a between-subject factor. Two-tailed effect sizes (i.e., Cohen's d), given the means, standard deviations, and sample sizes per condition, were used to test differences between conditions regarding interest and perceived social presence. Effect sizes were divided into the following five levels: trivial (Cohen's d≤.2), small (>.2), moderate (>.5), large (>.8), and very large (>1.3) [42]. BODY.RESULTS: Participants in the experimental condition focused on the social presence elements, both in terms of frequency (F(1, 98) = 40.34, p<.001) and duration (F(1, 88) = 39.99, p<.001), while participants in the control condition paid hardly any attention to these lookzones. There were, however, no differences between conditions in terms of frequency (F(1, 101) = 1.59, p = .21) and duration (F(1, 90) = 0.13, p = .72) of fixations on the main text. Figures 2 and 3 indicate that participants in the experimental condition were attracted by the social presence elements, but this did not affect their attention to the main text. Participants in the experimental condition scored higher on interest (M = 4.7 vs. 4.2, Cohen's d = 0.32) and perceived social presence (M = 4.6 vs. 3.1, Cohen's d = 1.22) in comparison with the control condition. These effects can be classified as small and very large respectively. 10.1371/journal.pone.0057067.g002Figure 2Number of fixations per page. 10.1371/journal.pone.0057067.g003Figure 3Total fixation duration (ms) per page. BODY.RESULTS.MAIN STUDY: The preparatory study demonstrated that the social presence elements attract participants' attention and increase perceived social presence, without decreasing attention for the main text. The aim of the main study was to test the proposed conceptual model and associated hypotheses. BODY.METHODS.MEASUREMENTS.HEPATITIS KNOWLEDGE QUESTIONNAIRE: Fifteen true/false items (including a 'don't know' option) about transmission, consequences, and prevention of HAV, HBV and HCV infections were used to assess Hepatitis knowledge. The sum score of all correctly answered items was used in the analyses. The correct answer to these items was available on the website. The items concerned practical knowledge and were used in a previous study [35]. BODY.METHODS.MEASUREMENTS.WEBSITE USE: Server registrations were used to assess website use [43], [44] which was operationalized by the number of pages visited (ranging from 0 to 12). Furthermore, time on the website was tracked to detect whether participants were simply clicking from one page to the other and therefore artificially boosting the number of pages visited. BODY.METHODS.MEASUREMENTS.USER PERCEPTIONS: Sets of three items each were used to measure efficiency (e.g., "I was able to access the information quickly on this website"; alpha = .97), effectiveness (e.g., "The website provided me with relevant information about ..."; alpha = .92), enjoyment (e.g., "I found my visit to this website enjoyable"; alpha = .97), and active trust (e.g., "I would act upon the information presented on this website if needed"; alpha = .89). Items were answered on a 7-point Likert scale ranging from (1) 'strongly disagree' to (7) 'strongly agree'. These measures were previously used and validated in the Dutch language [24]. Similar to the preparatory study, two items were used to measure interest (alpha = .82). BODY.ANALYSES: First, independent samples t-tests and chi-square tests were conducted to test whether there was selective dropout or differences in dropout between conditions. Subsequently, independent-samples t-test were conducted to test for differences between conditions in terms of perceived social presence and both measures of website use. For this analysis, time on website was log-transformed to meet the assumption of normality. A repeated measures analysis of variance (ANOVA) was conducted to test for differences in Hepatitis knowledge between conditions over time. Multivariate ANOVAs were conducted to test for differences in user perceptions between conditions. All these analyses were conducted using Predictive Analytics SoftWare Statistics 18.0 (International Business Machines Corporation, Armonk, NY). Second, using Mplus 5 (Muthén & Muthén, Los Angeles, CA), structural equation models (SEMs) using all available data were constructed to test the hypothesized conceptual model. Website use—a latent construct made up from number of pages visited and time on the website—was regressed on efficiency, effectiveness, enjoyment, interest, and active trust, which were all latent constructs using the items described above. Active trust was regressed on efficiency, effectiveness, and enjoyment. Effectiveness, enjoyment, and interest were regressed on social presence. Subsequently, (1) non-significant paths were left out of the conceptual model for the sake of parsimony, and (2) additional paths were added to the conceptual model based on significant modification indices. The latter was done to explore whether unanticipated relationships might explain variance in website use (which was not the case). A level of significance of p<.05 was used for the relationships within the model. Model fit indices used were the comparative fit index (CFI), the Tucker-Lewis index (TLI), the root mean square error of approximation (RMSEA), and the standardized root mean square residual (SRMR). Both CFI and TLI are goodness-of-fit indices where larger values signal better fit. Values over .95 indicate close fit. The RMSEA and SRMR are goodness-of-fit indices where larger values signal worse fit. Indicators of close fit are, respectively, RMSEA ≤.05 and SRMR ≤.09 [45], [46]. BODY.RESULTS.PARTICIPANTS: Of those 982 potential participants that were invited, 458 participated in the study (47%). Half of the participants were female and the mean age of participants was 49 years (SD = 16). Of the participants, 30% had a low level of highest completed education (equivalent to primary school/junior high school), 38% an intermediate level (equivalent to senior high school/junior college), and 32% a high level (equivalent to college/university). Those 458 that participated were invited to complete the follow-up measure and 402 of them did so (88%). There was no selective dropout regarding gender (χ2 (1, N = 458) = 0.3, p = .57), level of education (χ2 = 5.0, p = .08), or Hepatitis knowledge at baseline (t(456) = 1.83, p = .07). Furthermore, dropout did not differ between the two conditions (χ2 (1, N = 458) = 3.2, p = .08). Those who dropped out, however, were younger (M = 43 vs 50 years, (t(456) = 3.19, p = .002). BODY.RESULTS.MANIPULATION CHECK: Participants in the experimental condition scored higher on perceived social presence than the control condition (M = 4.9 vs. 4.4, t(456) = 4.11, p<.001, Cohen's d = 0.38), which indicates that the manipulation did succeed. This effect can be classified as small. BODY.RESULTS.WEBSITE USE AND HEPATITIS KNOWLEDGE: Participants in the experimental condition did not differ in website use in comparison with the control condition; neither in terms of the number of pages visited, nor in terms of time on the website (Table 1). Effect sizes were trivial. Thus, there is no support for Hypothesis 1. The increase in Hepatitis knowledge between pre-test and post-test did not differ between both conditions (F(1, 400) = 0.72, p = .40), but there was a significant increase regarding Hepatitis knowledge between pre-test and post-test across conditions (M = 6.4 vs. 8.3, F(1,400) = 172.98, p<.001, Cohen's d = 0.65), which can be defined as a moderate effect size. 10.1371/journal.pone.0057067.t001 Table 1 Differences in website use between conditions. Variable Experimental condition Control condition t p Cohen's d M SD M SD Number of pages (0–12) 7.5 4.0 8.0 3.8 1.44 .15 0.14 Time on website (sec) 151.0 117.4 150.8 107.8 0.01 .99 0.00 BODY.RESULTS.USER PERCEPTIONS: There were no differences between conditions in terms of efficiency (F(1, 456) = 0.84, p = .36), effectiveness (F(1, 456) = 0.03, p = .87), enjoyment (F(1, 456) = 0.08, p = .78), active trust (F(1, 456) = 0.03, p = .87) and interest (F(1, 456) = 0.07, p = .80). Figure 5 illustrates the final structural equation model testing the associations between user perceptions and website use across conditions, because there were no differences on both measures of website use. The final model is mostly in line with Hypothesis 2: Efficiency (M = 6.5, SD = 0.84), effectiveness (M = 6.4, SD = 0.87), and enjoyment (M = 5.6, SD = 1.21) were positively associated with website use and these associations were partly mediated by active trust (M = 5.8, SD = 1.23). The association between efficiency and website use, however, was fully mediated by active trust. Since there were no differences between conditions in terms of website use, there is neither support for Hypothesis 3a nor for Hypothesis 3b. Interest (M = 5.9, SD = 1.06), however, was positively associated with and contributed to explaining variance in website use besides efficiency, effectiveness, enjoyment, and active trust. The explained variance (R2) of the number of pages visited and time on the website is .63 and .51 respectively. The CFI and TLI are .98 and .97, respectively; RMSEA and SRMR are .06 and .04, respectively. All of these fit indices indicate a close fit for the final model. 10.1371/journal.pone.0057067.g005Figure 5Final model including standardized betas of significant paths within the model. BODY.DISCUSSION: The key finding of the current study is that adding social presence elements did not affect actual use of an intervention website within a public health context. A possible explanation for this lack of effect can be derived from the results of the preparatory study: Even though the social presence elements attract attention, the frequency and duration of fixations on these elements was limited in comparison with the fixations on the main text. If the findings of the preparatory study are extrapolated to the main study, then this might explain the limited impact of social presence elements on actual website use. It could be that people on a health-related website are mainly interested in the actual information it provides (i.e., the main text instead of social presence elements), which is mostly related to the utilitarian value of a website [47]. To date, however, the use of social presence elements has only been tested on more hedonically oriented websites (such as shopping for clothing) [14], [48]. It might be that social presence is more important on hedonically oriented websites in comparison with health-related websites, which are more utilitarian in nature. Even within the e-commerce context, there are differences between products: A previous study compared clothing (a more hedonic product for which consumers seek fun and entertaining shopping experiences) and headphones (a more utilitarian product for which consumers primarily seek detailed product information). Unlike clothing, higher levels of social presence on websites selling headphones did not have a positive effect on attitudinal antecedents [49]. In line with this, Nusair, Yoon, and Parsa [50] found that motivation variables, utilitarian and hedonic, moderate the effect of the web quality dimensions on satisfaction. There are two other points that need to be raised while interpreting the findings of this study. First, there was a difference in effect size regarding perceived social presence, which was very large in the preparatory study, but only small in the main study. A speculative explanation could be that the sample of the preparatory study was much younger and might therefore be more open to social presence elements on websites. It is in our opinion more likely, however, that this is indicative of the difference between the lab setting in which participants had to look at the website in a more controlled environment versus the main study in which a real-life situation was mimicked. In the latter case, participants might pay only limited attention to a website [51], especially when added elements increase its complexity [33]. Second, this is the first study to investigate the effect of adding social presence elements on actual use instead of only intention to use or attitude towards a website. Although intention and attitude are important predictors of behavior [15], the intention-behavior gap is a well-known phenomenon in behavioral research: intentions account for 28% of the variance in behavior in prospective studies [52]. The explained variance of both behavioral measures in the current study (i.e., the number of pages visited and time on the website) was high and there was a close fit for the final model. In other words, user perceptions are appropriate predictors for explaining use of an intervention website in a public health context. Moreover, the relationships between these user perceptions and website use are comparable to the few other studies that are available within the field of attrition science regarding Internet-delivered interventions [24], [25]. The current study extends previous studies by including a measure of interest and demonstrates its added value besides the other perceptions. This is in line with the theoretical assumption of Silvia [29] that interest and enjoyment differ in critical ways and contributes to highly needed theory development within the field of attrition science [3]. A possible limitation of the study at hand is that participants in the main study were highly interested in the subject (i.e., Hepatitis), since they agree to participate in a study about this topic. Although participants perceived the website to be interesting (after being exposed to it), the low scores regarding Hepatitis knowledge at pre-test do not provide support for a possible selection bias regarding participants that were highly interested in the subject before the study. The increase regarding Hepatitis knowledge one week later is very positive, especially since participants were not necessarily looking for information on Hepatitis. The latter is essential in primary prevention websites aimed at the general public. Future research needs to focus on characteristics of social presence elements that might affect their impact. The human images of people in the current study, for example, varied in age and gender. A previous study on Product Recommendation Agents (PRA; a decision support system that helps consumers to gather, screen, and evaluate available product information on the Internet), however, found that ethnicity-matched PRAs scored higher on perceived social presence, enjoyment, and effectiveness [53]. Another study revealed that PRAs with higher personalization also elicited more trust [54]. Personalization is one of the strategies, besides feedback and content matching, that is part of tailoring: creating communications individualized for their receivers, with the expectation that this individualization will lead to larger intended effects of these communications [55]. It might be that social presence elements on intervention websites in a public health context need to be tailored – a commonly used and effective method for the actual information on intervention websites [56]. Furthermore, the website used in this study was limited to providing knowledge about Hepatitis. It is worthwhile to investigate whether social presence elements may be more effective in increasing website use regarding intervention websites that are aimed at demonstrating and practicing skills (e.g., using role models) or reducing stigma that may be related to health behavior. Another possible avenue for future research is whether or not cultural differences might be in play. Based on Hofstede's cultural value dimensions [57], a previous study revealed that the impact of pleasure on online behavior was higher for Canadian website visitors, while the impact of dominance was higher for Chinese website visitors [58]. To our knowledge, only one study has made a cross-cultural comparison of the impact of social presence elements, but this was limited to the impact user perceptions and actual use was not taken into account [59]. Hence, this is a nascent area worthy of additional investigation.

TITLE: Comparative utility of disability progression measures in PPMS ABSTRACT.OBJECTIVE:: To assess the comparative utility of disability progression measures in primary progressive MS (PPMS) using the PROMiSe trial data set. ABSTRACT.METHODS:: Data for patients randomized to placebo (n = 316) in the PROMiSe trial were included in this analysis. Disability was assessed using change in single (Expanded Disability Status Scale [EDSS], timed 25-foot walk [T25FW], and 9-hole peg test [9HPT]) and composite disability measures (EDSS/T25FW, EDSS/9HPT, and EDSS/T25FW/9HPT). Cumulative and cross-sectional unconfirmed disability progression (UDP) and confirmed disability progression (CDP; sustained for 3 months) rates were assessed at 12 and 24 months. ABSTRACT.RESULTS:: CDP rates defined by a ≥20% increase in T25FW were higher than those defined by EDSS score at 12 and 24 months. CDP rates defined by T25FW or EDSS score were higher than those defined by 9HPT score. The 3-part composite measure was associated with more CDP events (41.4% and 63.9% of patients at 12 and 24 months, respectively) than the 2-part measure (EDSS/T25FW [38.5% and 59.5%, respectively]) and any single measure. Cumulative UDP and CDP rates were higher than cross-sectional rates. ABSTRACT.CONCLUSIONS:: The T25FW or composite measures of disability may be more sensitive to disability progression in patients with PPMS and should be considered as the primary endpoint for future studies of new therapies. CDP may be the preferred measure in classic randomized controlled trials in which cumulative disability progression rates are evaluated; UDP may be feasible for cross-sectional studies. BODY: Patients with primary progressive MS (PPMS) represent 10%–15% of patients with MS and suffer the highest neurodegeneration-related disability.1–4 There are currently no approved treatments to ameliorate disease progression in PPMS, and there are several barriers to developing therapeutics in progressive MS.2,5 Specifically, individual disability measures may lack the sensitivity required to reveal all cases of disease progression within the defined time frame of a clinical trial.6 The Expanded Disability Status Scale (EDSS) remains the typical outcome measure in PPMS trials. However, it has been widely recognized as inadequate7 and is associated with several important weaknesses.3 Other widely used measures of disability in MS include the timed 25-foot walk (T25FW) and the 9-hole peg test (9HPT).8,9 Recent studies have suggested that a combined disability measure that incorporates scoring from multiple instruments—for example, using T25FW, 9HPT, and EDSS—may be associated with more progression events among patients with PPMS, and thus higher reported event rates than achieved with any single instrument alone.9,10 The PROMiSe study represents one of the best and largest (N = 943) data sources on patients with PPMS and offers a unique opportunity to assess the value of different functional measures of disability progression in a clinical trial setting.11 We report here the results of an analysis of the PROMiSe data set undertaken to better understand the utility of the different measures of disability progression—EDSS, T25FW, and 9HPT alone and in combinations—and to inform the design of future trials in PPMS. BODY.METHODS.STANDARD PROTOCOL APPROVALS, REGISTRATIONS, AND PATIENT CONSENTS.: Participating study sites in each individual country locally ensured all necessary regulatory approvals (e.g., institutional review boards/institutional ethics committees) in accordance with local regulations, including local data protection regulations used in the original collection of the data for this secondary data analysis. BODY.METHODS.STUDY DESIGN.: The data set for this analysis was derived from the PROMiSe study, a randomized, double-blind, placebo-controlled clinical trial with a planned duration of 3 years.11 The design of the PROMiSe study has been reported previously.11 In summary, patients who took part in the PROMiSe study (N = 943) were randomized in a 2:1 ratio to receive glatiramer acetate (GA) 20 mg once daily (n = 627) or identical-appearing placebo (n = 316) by daily subcutaneous injection.11 The primary endpoint was time to confirmed disability progression (CDP), with CDP being defined as a change of ≥1 point on the EDSS sustained for 3 months in patients with a baseline EDSS score of 3.0–5.0, or a change of ≥0.5 points for 3 months in those with a baseline score of 5.5–6.5.11 Patients also completed the MS Functional Composite (MSFC) evaluation, a 3-part test including the T25FW and 9HPT and the Paced Auditory Serial Addition Test (PASAT).11 BODY.METHODS.PATIENTS.: Patients eligible to participate in the PROMiSe study were aged between 30 and 65 years, with an EDSS score of 3.0–6.5, investigator-confirmed PPMS, and progressive symptoms including myelopathy for ≥6 months before screening. Additional eligibility criteria included evidence of pyramidal damage on neurologic examination, including a pyramidal functional system score ≥2, and evidence of multilevel CNS disease.11 Patients with any history of MS relapse, those with lymphopenia <3,000 cells/mL, and those who had used an interferon-β drug, immunosuppressant, immunomodulating agent, corticosteroid, or investigational drug within 3 months of study initiation were not eligible to take part.11 Only data for patients randomized to placebo in the PROMiSe trial were included in this analysis in order to compare clinical measures in patients with PPMS for whom the natural course of the disease was not potentially affected by exposure to GA treatment. BODY.METHODS.OUTCOME EVALUATIONS.: Disability was assessed using change in single (EDSS, T25FW, and 9HPT) and composite disability measures. Combination endpoints were defined as progression by EDSS or T25FW, 9HPT or T25FW, or by EDSS or T25FW or 9HPT. The PASAT was not included in this analysis of the PROMiSe database because of the potential for practice effects occurring from repeated administration. Moreover, the test may have limited measurement reliability, is sometimes difficult to administer, has been used less frequently in recent MS trials, and is not anticipated to be featured in future trials. As per the PROMiSe study protocol, worsening of the EDSS was defined as a change from baseline of ≥1 point (for baseline EDSS 3.0–5.0) or ≥0.5 points (for baseline EDSS 5.5–6.5).11 Worsening of the T25FW was defined as an increase of ≥20% compared with baseline or as the inability to complete the T25FW after baseline due to worsening disability (increase of EDSS to 7.0 or higher).12 Worsening of the 9HPT was defined as an increase of ≥20% compared with baseline (calculated as the average across 4 trials: 2 in the dominant hand and 2 in the nondominant hand) or as the inability to complete the 9HPT after baseline due to worsening disability.12 For each measure, rates of unconfirmed disability progression (UDP) and of 3-month CDP were determined. Two trial methodologies were evaluated: cumulative and cross-sectional. The cumulative approach describes the design of the PROMiSe trial and similar large-scale randomized clinical trials, with outcome measurement every 3 months and disability progression at any time point during follow-up contributing to an overall CDP proportion. The cross-sectional approach describes the design for simple, straightforward investigator-initiated trials such as futility trials13 and observational studies on PPMS disability progression.10 In this case, the measurement at one follow-up time point (e.g., 12 months) is compared with the baseline measurement. This analysis is restricted to patients in the PROMiSe trial receiving placebo in order to compare clinical measures in patients with PPMS for whom the natural course of the disease was not affected by exposure to GA treatment. BODY.METHODS.STATISTICAL ANALYSES.: Cumulative progression rates were estimated using the Kaplan-Meier methodology. For summaries by month, 1 month was defined as 28 days. For confirmed disability events, patients who experienced a worsening event in the measure of interest that could not be confirmed due to the patient's discontinuation from the trial prior to the confirmation visit were censored at the time of the worsening. Other patients who did not experience the progression event were censored at their last day in the study. Cross-sectional progression rates were determined by comparing baseline and follow-up disability measures. Patients missing any of the 3 disability measures at baseline, the time point of interest, or the corresponding confirmation assessment were excluded from the analysis. The relative percentage contribution of each disability measure to disease progression rates observed for the combined EDSS or T25FW or 9HPT measure was derived by determining the number of individual patients progressing on 1, 2, or all 3 outcomes measures. BODY.RESULTS: A total of 316 patients were assigned to receive placebo in the PROMiSe study; 314 of these patients had a postbaseline EDSS assessment, and these patients constituted the population for the cumulative analyses presented here. Of these, 272 patients were included in the 12-month cross-sectional analysis, and 214 patients constitute the population for the 24-month cross-sectional analysis. Baseline demographic and clinical characteristics for the 314 patients who received placebo are shown in table 1.11 The baseline characteristics of the subgroup of patients assigned to receive placebo were consistent with those of the entire PROMiSe patient population.11 Table 1 Baseline demographic and clinical characteristics of patients who took part in the PROMiSe trial and were randomized to receive placebo Table 2 shows cumulative and cross-sectional UDP and CDP rates at 12 and 24 months. Among single measures, a ≥20% worsening in T25FW (T25FW 20%) was associated with the highest cumulative progression rates, followed by EDSS and then a ≥20% worsening in 9HPT (9HPT 20%) at both the 12- and 24-month time points. Among combined measures, the composite of all 3 individual measures (i.e., EDSS or T25FW 20% or 9HPT 20%) had the highest cumulative 3-month CDP rate at both 12 and 24 months (41.4% and 63.9%, respectively). Cross-sectional 3-month CDP rates were also highest for the composite of all 3 scores than for any single or dual combination of scores at both 12 and 24 months (37.5% and 57.5%, respectively). UDP proportions were consistently higher than CDP proportions for all measures, with a considerably greater difference between UDP and CDP in the cumulative incidence of the composite of all 3 individual measures (1 year: 63.8% and 41.4%, respectively; 2 years: 82.1% and 63.9%, respectively) compared with the cross-sectional proportions (1 year: 47.8% and 37.5%, respectively; 2 years: 57.5% and 52.8%, respectively). Table 2 Cumulative and cross-sectional PROMiSe disease progression rates at 12 and 24 months The proportion of patients with UDP and 3-month CDP for each of the disability metrics over the course of the study is shown in figure 1 for each study paradigm (cumulative and cross sectional). The T25FW 20%-alone values were closely aligned with those from the combined EDSS-or-T25FW 20% values at all time points over the course of the study (table 3). Figure 1Comparison of cumulative and cross-sectional UDP and CDP rates for EDSS, T25FW 20%, and 9HPT 20%(A) EDSS rates. (B) T25FW 20% rates. (C) 9HPT 20% rates. 3M CDP = 3-month confirmed disability progression; 9HPT = 9-hole peg test; EDSS = Expanded Disability Status Scale; T25FW = timed 25-foot walk; UDP = unconfirmed disability progression. Table 3 Disease progression rates over the course of the PROMiSe study The relative contribution of each of the individual component measures—together and in different combinations—of a 3-part composite measure (EDSS or T25FW 20% or 9HPT 20%) is shown in figure 2 and figure e-1 at Neurology.org/nn. T25FW 20% had the greatest contribution to the overall measure, followed by EDSS, and then 9HPT 20%. These figures support the notion that CDP as defined by T25FW identifies individuals as progressors that the other measures would not have been identified. In other words, a greater proportion of patients identified as progressors by T25FW were not identified as progressors by EDSS or 9HPT, as compared with the proportion of patients identified as progressors by EDSS who were not identified as progressors by T25FW or 9HPT (figure e-2). Figure 2Contribution of individual clinical measures evaluating patient worsening at 24 months (cross-sectional 3-month CDP) within a 3-part combined measure (EDSS or T25FW 20% or 9HPT 20%)(A) Cumulative UDP. (B) Cumulative 3-month CDP. (C) Cross-sectional UDP. (D) Cross-sectional 3-month CDP. 3M CDP = 3-month confirmed disability progression; 9HPT = 9-hole peg test; EDSS = Expanded Disability Status Scale; T25FW = timed 25-foot walk; UDP = unconfirmed disability progression. BODY.DISCUSSION: The data presented here show that among patients with PPMS randomized to receive placebo in the PROMiSe study, T25FW 20% at 12 and 24 months was associated with higher rates of disability progression than EDSS, and both T25FW 20% and EDSS were associated with higher disability rates than 9HPT 20%. The 3-part combination measure (EDSS or T25FW 20% or 9HPT 20%) was associated with more 3-month CDP events (41.4% and 63.9% of patients as 12 and 24 months, respectively) than the 2-part measure (EDSS or T25FW 20% [38.5% and 59.5%, respectively]), and more progression events than any single measure (EDSS, 14.9% and 32.4%, respectively; T25FW, 33.5% and 52.0%, respectively; 9HPT, 8.4% and 18.0%, respectively). Minimal changes in the 9HPT 20% outcome were noted over the course of the PROMiSe trial. As such, this measure may be less suitable than EDSS and T25FW 20% when used alone; however, 9HPT may still have utility as part of a composite measure. Moreover, future studies should explore thresholds other than 20% worsening on performance measures such as 9HPT. A marked difference was noted between cumulative and cross-sectional progression rates, which may inform the design of PPMS trials. Cumulative progression rates were generally higher than cross-sectional rates, but more strikingly, there was a greater difference between unconfirmed and CDP using the cumulative approach. Based on our results, the normative approach to use CDP in classic randomized controlled trials is supported. Failure to confirm disability progression may have a neurobiological basis or may be due to measurement unreliability; it would be preferable to exclude these from pivotal trial analyses in both cases. For trials using the cross-sectional approach, measures of UDP may still be feasible. Such studies would be resource-sparing and less burdensome for participants, but would have acknowledged data sparsity and methodological limitations. The observations presented here add to a growing body of evidence that suggests that combining specific disability measurements may be more useful in the clinical trial setting for assessing progression in patients with PPMS than single measures.9,10,14–16 In a retrospective database study of 181 patients with progressive MS (primary 47% of patients and secondary 53% of patients), combining change in T25FW with EDSS was significantly more predictive of patient prognosis than EDSS alone.15 An earlier retrospective database study of 161 patients with PPMS found that when comparing single measures or 2-instrument combinations involving EDSS, T25FW, and 9HPT, T25FW/9HPT predicted the greatest number of progression events after 1 year (46% of patients progressed compared with 17%, 34%, and 20% for the individual measures, respectively), and the combination of T25FW/EDSS predicted the greatest number of progression events after the second year (57% of patients progressed compared with 32%, 46%, and 24% for the individual measures, respectively).11 An evaluation of data from the 96-week Olympus (rituximab) trial17 in the 147 patients with PPMS randomized to placebo found that a combined measure consisting of EDSS/T25FW/9HPT was associated with more CDP events than did any single or dual combination measure.11 Progression rates at 96 weeks were 61.9% and 54.8% for those patients with an initial confirmed progression event at 12 and 24 weeks, respectively.9 Confirmed progression rates for EDSS alone were 38.5% and 30.4%; for T25FW alone the rates were 51.0% and 44.5%; and for the 9HPT alone the rates were 21.1% and 17.4%, respectively.9 Most recently, the large, prospective INFORMS (fingolimod) study, comprising patients with PPMS treated for at least 3 years, reported that a composite of EDSS, T25FW, or 9HPT predicted more progression events (80.3% cumulative probability of CDP) than any of the individual components alone (EDSS 58.7%, T25FW 70.0%, and 9HPT 41.3% of patients) among those randomized to placebo.14 Although those previous published studies offer similar conclusions to this study regarding the relative utility of outcome measures, they differ in several important ways that distinguish them from our analysis of the PROMiSe data set. The retrospective database study included both primary and secondary progressive patients with MS, was primarily focused on outcomes of early vs late changes on clinical scales, included patients who were exposed to disease-modifying therapies (DMTs) during the course of the study, did not set a minimum disability criterion as per EDSS for study entry, and included less-frequent patient assessment.15 Similarly, the earlier study likely included patients exposed to DMTs, while its entry criteria included an EDSS threshold score of 2.0–6.5, representing a less-disabled patient population than that of the PROMiSe study.10 The same EDSS criteria applied to the Olympus study, whereas none of these 3 studies, nor the INFORMS study, compared confirmed with unconfirmed progression or offered a cross-sectional analysis of disease progression data.9,14 Two other recently published studies address similar themes and arrive at compatible conclusions to those of this study.18,19 In one of these studies, however, the patient population consisted entirely of those with secondary progressive MS,18 whereas the other had a lower EDSS inclusion threshold (1–7), allowed inclusion of patients who had been treated with DMTs as recently as 3 months prior to study entry, and was intended as a validation study of a novel disability score (CombiWISE); that scoring measure, in addition to EDSS, T25FW, and 9HPT, also includes the Scripps Neurological Rating Scale.19 And, as with the previously noted studies, neither unconfirmed disease nor a cross-sectional analysis was included in the design of these studies.18,19 Thus, this study offers several unique features that make it a potentially valuable contribution to the literature, such as reflecting the untreated natural history of PPMS by including only patients receiving placebo, including patients with more severe disability, comparing confirmed and unconfirmed disease progression, and including a cross-sectional analysis. There is an unmet need for controlling disease progression in patients with PPMS. For these patients, individual measures of disease progression may limit the potential to assess the benefit of new agents to the extent that the composite measures have higher event rates. Based on the observations presented here and those from previous studies, future studies of agents for the treatment of PPMS may benefit from either using T25FW 20% as a single outcome measure or using T25FW 20% in combination with the EDSS as a primary efficacy endpoint rather than EDSS alone, particularly if higher event rates over shorter exposure periods are sought. It is largely unknown whether specific disability outcomes would differentially detect treatment effects or whether this would differ according to the mechanism of the treatment under study. BODY.SUPPLEMENTARY MATERIAL: Data Supplement

TITLE: Clinical efficacy of ABSTRACT: Aging has become one of the distinctive demographic phenomena in the 21st century and its social, economic and health implications are the most challenging issues. Senile Memory Impairment is a common condition characterized by mild symptoms of cognitive decline and occurs as a part of the normal aging process. It can be correlated to "Jarajanya Smrtibhramsha" according to Ayurveda. The present study deals with the efficacy of Guduchyadi Medhya Rasayana on Senile Memory Impairment. A total of 138 patients aged in between 55–75 years were registered and randomly divided into two groups as the trial and control groups. The drugs were administered for 3. The trial drug showed memory enhancement, anti-stress, anti-depressant and anxiolytic properties. The trial group showed better results in the management compared to the control group. BODY.INTRODUCTION: Aging has become a great issue for many countries due to a worldwide life prolongation. Number of people over 60 years has grown rapidly to 10% of the world population.[1] In India too, in the last decade, increasing population aged has been projected significantly.[2] So, it is associated with physiological, functional, and pathological changes affecting physical, psychological, emotional and social well-being.[3] The major consequences of the increasing number of individuals in advanced age group is the increase in number of patients suffering from age-related disorders. Conservative estimation revealed that 25% of the elderly persons have significant psychiatric symptoms[4] and the number of psychologicaly ill elderly persons was estimated to be 9 million in the year 2005.[5] Though psychological disorders are common among the aged, it frequently remains undetected and untreated. It induces functional disability, disturbs rehabilitation, burdens the health system and impairs the quality of life of elderly individuals.[6] Research based on the psychological health revealed that age-associated cognitive like memory disorders are found in high prevalence among the aged.[7] Subjective complaints from individuals with age-associated memory impairment often include difficulties in remembering names and words, etc. So, this condition is the result of physiological changes in the aging brain and not a specific neurological disorder.[8] In Ayurveda literature, impairment of memory is mentioned as Smritibhramsha which occurs due to vitiation of Rajas and Tamas Doshas in the mind.[9] The appearance of Jara (aging) at the appropriate age is termed as "Kalaja Jara",[10] and memory impairment appears as a clinical Rupa (feature) of Jara,[11] hence Senile Memory Impairment can be correlated as Jarajanya Smritibhramsha according to Ayurveda. In Sarangadhara Samhita, it is clearly emphasized that Medha (intellect) and Buddhi (wisdom) deteriorate in the fourth and ninth decades of life accordingly.[12] Both are closely associated with the mind and help for the normal function of the mind. If these components decrease in the mind, then it will affect the normal function of mind. Moreover, Caraka has noted that cognitive functions are thoroughly affected in old age as Grahana (power of understanding), Dharana (power of retention), Smarana (power of memorizing), Vachana (speech) and functions of sense organs qualitatively.[13] BODY.INTRODUCTION.IMPORTANCE OF : The aging process occurs over all the body. It does not occur in the same way in every human being. People's lifestyles also have additional influence on aging.[14] The decline in brain weight and volume, degeneration of intracellular organelles, changes in cellular DNA and RNA, neuronal degeneration as well as significant loss of synapses are the salient features of aging in the brain.[15] Therefore, psychological and neurological manifestations often form the hallmark of senility. Hence, Ayurveda clearly emphasizes that in the fourth and ninth decades of lifespan, individuals are subjected to the loss of Medha (intellect) and Buddhi (wisdom). Hence, it seems therapeutic measures of Medhya Rasayanas are exclusively needed for elders because they are brain tonics and improve the psychological faculties. It is responsible for Dhi (intelligence), Dhrti (retention power), Smriti (memory) and also possesses anti-stress and adaptogenic effect.[16] Having considered all literary views and ideas as above mentioned, Guduchyadi Medhya Rasayana (GMR)[17] has been attempted to evaluate its efficacy on Senile Memory Impairment. BODY.MATERIALS AND METHODS.SELECTION OF THE PATIENTS: Total 138 patients having symptoms of memory impairment attending O.P.D. and I.P.D. of Department of Kaya Chikitsa of I.P.G.T. and R.A., Gujarat Ayurved University, Jamnagar were selected for the present study. A purposive sampling technique was adopted irrespective of their sex, religion, occupation, educational status, etc. BODY.MATERIALS AND METHODS.CRITERIA OF INCLUSION: Patients aged 55 and above up to75 years of age.Complaints of memory impairment on the basis of short term and long term. Short term, e.g. misplacing objects, difficulty in remembering multiple items to be purchased, difficulty in recalling information quickly, problems in remembering names and telephone numbers.Long term, e.g. difficulty in recall of special events of personal life, difficulty in recall of previous histories of life and recognition.Patients suffering from dementia. BODY.MATERIALS AND METHODS.CRITERIA OF EXCLUSION: Patients below 55 years and above 75 years of age.Patients with evidence of delirium, confusion or other disturbances of consciousness, Parkinson's disease, stroke, intracranial hemorrhage, brain tumors, history of alcoholism or drug dependence, use of any psychotropic drug, Alzheimer's disease, diabetes mellitus, etc. BODY.MATERIALS AND METHODS.INVESTIGATIONS: Routine hematological and biochemical tests were carried out before and after the treatment. Among the biochemical tests, serum Acetylcholine Esterase (AChE) estimation, as a biomarker of Senile Memory Impairment, was carried out. BODY.MATERIALS AND METHODS.PLAN OF THE STUDY: Patients fulfilling the inclusion criterion were grouped in to two after obtaining written consent. BODY.MATERIALS AND METHODS.GROUP A (TRIAL GROUP): Patients of this group were administered with the Granules of Guduchyadi Medhya Rasayana (GMR), which contains Guduchi (Tinospora cordifolia Wild.), Apamarga (Achyranthes aspera Linn.), Vidanga (Embelia ribes Burm. f.), Shankhapushpi (Convolvulus pluricaulis Chois.), Vaca (Acorus calamus Linn.), Haritaki (Terminalia chebula Zetz.), Kushtha (Saussurea lappa C.B. Clarke), Shatavari (Asparagus racemosus Wild.), Cow's ghee and sugar. Dosage: 5 g, 3 times per day, after meal Anupana: Water in sufficient quantity Duration: 3 months Total, 73 patients were registered. BODY.MATERIALS AND METHODS.GROUP B (CONTROL GROUP): Patients of this group were administered Sarkaradi Granules (SG). Dosage: 3 g, 3 times per day, after meal Anupana: Water in sufficient quantity Duration: 3 months Total 65 patients were registered.. Both the drugs were prepared at Pharmacy, Gujarat Ayurved University, Jamnagar. BODY.MATERIALS AND METHODS.CRITERIA OF ASSESSMENT: Effects of Medhya Rasayana on memory impairment were assessed in two ways, i.e. as subjective and objective, before and after the treatment. After completion of the treatment, the patients were graded into five groups based on their improvement in order to ascertain the overall therapeutic efficacy. The data so obtained were analyzed using SPSS and Sigma Stat statistical packages. Observations and results pertaining to clinical parameters were presented in tabular form along with the findings of statistical analysis. Paired "t" test was adopted to find out the effect of therapy for each group individually, while unpaired "t" test used for comparative effect. BODY.MATERIALS AND METHODS.CRITERIA OF ASSESSMENT.SUBJECTIVE CRITERIA: Assessments on improvements in signs and symptoms were carried out using appropriately designed scoring method, with the scores ranging from 0 to 4. Cognitive state was assessed by Mini Mental State Examination.[18] and psychological health of the patients was evaluated with Hamilton Rating Scales for Depression and Anxiety.[19] BODY.MATERIALS AND METHODS.CRITERIA OF ASSESSMENT.OBJECTIVE CRITERIA: Serum AChE estimation was carried out as a biomarker of Senile Memory Impairment. BODY.OBSERVATIONS AND RESULTS: Total 138 patients of Senile Memory Impairment were registered in the study. Forty-three patients in the Group A (trial group) and 56 patients in the Group B (control group) completed the treatment, while 30 patients in Group A and 9 patients in Group B discontinued. In this study, various etiological factors were identified. Maximum number of patients reported Chinta (thoughts; 92%), followed by Krodha (anger; 83.3%), Shoka (worries; 70.2%), Chittodvega (anxiety; 64.4%), Bhaya (fear; 51.4%), Ruksha Ahara (dried food; 55.7%), Rattri Jagarana (awaking at night; 87%), etc. BODY.OBSERVATIONS AND RESULTS.EFFECT OF THERAPY: Both the groups showed highly significant (P`0.001) relief from all the chief complaints [Table 1 and Table 2]. Table 1 Effect of GMR in Group A on the symptoms of Senile Memory Impairment Table 2 Effect of SG in Group B on the symptoms of Senile Memory Impairment In comparison to Group B, Group A showed highly significant relief in symptoms such as names forgotten (63.56%), numbers forgotten (61.62%), and memory impairment in recalling special events in life (59.71%) at a level of P`0.001, and forgetfulness (68.60%), losing of valuables (73.64%), difficulty in remembering multiple items to be purchased (68.21%), inability to concentrate (68.14%), impaired recognition (79.7%), memory impairment of recall of previous histories (60.66%) at a level of P`0.01, and a significant relief (P`0.05) from difficulty in recalling information quickly (71.57%), depression (89.66%), and inability to relax (82.93%) [Table 3]. Table 3 Comparison of the effects of GMR and SG on the symptoms of Senile Memory Impairment With respect to Dosha Dushti, the Group A showed significant relief from Vata Dushti (85.48%), Pitta Dushti (94.15%) and in Kapha Dushti (84.3%), while the Group B showed significant relief from Vata Dushti (68.3%), Pitta Dushti (61.78%) and in Kapha Dushti (62.75%). On Hamilton Anxiety Rating Scale, both the groups showed highly significant relief (P`0.001) from all the symptoms individually. In comparison to the results obtained from Group B, the Group A showed highly significant relief with respect to intellectual cognitive functions (79.03%), somatic (muscular) disorders (83.71%), depression (74.12%), and a significant relief from fear (81.11%). On Hamilton Depression Rating Scale, both the groups showed highly significant relief (P`0.001) from all the symptoms individually. However, the comparative results were insignificant. The results of symptoms of Mini Mental State Examination showed that both the groups provided highly significant (P`0.001) improvement individually. In comparison with SG, GMR showed highly significant improvement in registration (9.30%), attention and calculation (24.49%), and a significant improvement in language (13.51%) and in total score (14.89%) [Table 4]. The results obtained with hematological parameters reveal that Group A showed [Table 5] highly significant increase in total RBC (1.20%) count, a significant increase in Hb% (1.34%), packed cell volume (PCV) (1.30%) and a significant decrease in erythrocyte sedimentation rate (ESR) value (7.40%), while Group B showed highly significant decrease in RBC count (4.30%), PCV (3.34%), and a significant decrease in Hb% (1.55%). Table 4 Comparison of the effects of GMR and SG on the symptoms of mini mental state examination Table 5 Effect of GMR on hematological parameters in Group A (n=43) With regard to the biochemical parameters, Group A showed highly significant decrease (P`0.01) in serum cholesterol, serum triglyceride, low density lipoprotein (LDL), and very low density lipoprotein (VLDL) levels, while high density lipoprotein (HDL) increased highly significantly (P`0.01) and a significant decrease (P`0.05) was found in blood urea (13.80%) as well as serum AChE (3.67%) within normal limits [Table 6]. The Group B showed highly significant increase in blood urea (10.04%), but it was within normal limit. Other parameters were found to be statistically insignificant. Table 6 Effect of GMR on biochemical parameters in Group A (n=43) Regarding the overall effect, Group A showed marked improvement in 18.6% patients, moderate improvement in 74.4% patients and mild improvement in 7.0% patients, while Group B showed moderate improvement in 32.2% patients and mild improvement in 67.8% patients. BODY.DISCUSSION: It has been mentioned in texts that GMR contributes to enhance the memory power within a short period. On the basis of Rasapanchaka, the pharmacotherapeutic properties reveal that it contains Katu, Tikta and Madhura in Rasa, and the effect of Katu and Madhura in Vipaka. Moreover, due to various properties of Tikshna, Laghu, Ruksha, Sara, Guru and Snigdha, it possesses markedly Ushna Virya and mildly Sheeta Virya. Accordingly, GMR was considered to be an effective treatment for Tridoshahara. In Ayurveda, it is clearly emphasized that Smritibhramsha (memory impairment) results due to Rajas and Tamasdoshas in the mind. These doshas are responsible for producing avarana over the mind. The relief from the symptoms has been found to be statistically highly significant. These observations reveal that the drug might have counteracted or pacified Raja and Tamo Doshas, so Avarana of mind was removed and the disease was subsided or controlled. Further pharmacological studies have proven that Vaca (Acorus calemus Linn.) and Shankhapushpi (Convolvulus pluricaulis Chois) have anxiolytic, memory enhancing, anti-stress, antidepressant, tranquilizing and sedative activities.[20, 21] Further, it was found that there was a significant improvement by 14.84% on the total score of the Mini Mental State Examination [Table 4]. So, these evidences indicate the positive role of Medhya property of GMR on Senile Memory Impairment among elderly individuals. Considering the symptoms of memory impairment along with the cognitive decline, a highly significant result was obtained both from GMR and SG [Table 1 and Table 2]. However, in comparison, GMR has given better relief for the majority of symptoms [Table 3]. The Medhya Rasayanas are special Ayurvedic neuro nutraceuticals which are specific to brain and nervous system. They are claimed to promote cognitive function of the brain.[22] Further, it was mentioned in the Caraka Samhita that Shankhapushpi, a Rasayana drug, has the property of promoting intellect.[23] Moreover, Group B also showed improvement of memory due to the Medhya effect of ghee.[24] The serum AChE value has shown an upper range in both the groups before the treatment and a significant decrease was observed after the treatment in Group A [Table 6]. The recent advances in clinical research reveal that AChE activity is increased in human cerebrospinal fluid during aging[25] and scientists believe that dementia-related memory issues stem from the increased breakdown of acetylcholine due to a preponderance of AChE which degrades acetylcholine.[26] Furthermore, a decrease in AChE activity has noted in the treated animals, leading to an increase in the cholinergic activity in the brain.[27] The significant reduction of AChE in Group A shows that GMR exerts its effect on memory enhancement by increasing the cholinergic function by preventing the breakdown of acetylcholine in the aging human brain. BODY.CONCLUSIONS: Senile Memory Impairment can be correlated to Jarajanya Smritibhramsha according to Ayurveda, and imbalance of Trigunas through aggravation of Rajas and Tamas Doshas in the mind leads to development of Senile Memory Impairment among elders. Modern literature reveals that Senile Memory Impairment is closely associated with cognitive decline during aging. All the patients of Senile Memory Impairment were having features of impairment of short memory and long memory in this study. Manasika Nidana (psychic factors), Aharaja (dietary) and Viharaja (behavioral) factors, which were reported by the patients, play a major role in the pathogenesis of Senile Memory Impairment by vitiating Rajas and Tamas Doshas in the mind. Those factors were Chinta (thoughts), Krodha (anger), Shoka (worries), Chittodvega (anxiety), Bhaya (fear), Moha (illusions), Ruksha Ahara (dried food), Ratri Jagarana (awaking at night), etc. GMR has shown statistically highly significant improvement on short memory impairment as well as long memory impairment in Senile Memory Impairment, at the level of P`0.001. Reduction of AChE showed enhancement of memory functions due to improvement of cholinergic actions in the human brain in the trial group (Group A). GMR has shown memory enhancing, antidepressant, anti-stress, and anxiolytic potential. Hence, Guduchyadi Medhya Rasayana therapy has proved so as to provide better improvement in comparison to the control group.

TITLE: Effect of ABSTRACT.BACKGROUND: To evaluate the value of KRAS codon 13 mutations in patients with advanced colorectal cancer (advanced CRC) treated with oxaliplatin and fluoropyrimidines. ABSTRACT.METHODS: Tumor specimens from 201 patients with advanced CRC from a randomized, phase III trial comparing oxaliplatin/5-FU vs. oxaliplatin/capecitabine were retrospectively analyzed for KRAS mutations. Mutation data were correlated to response data (Overall response rate, ORR), progression-free survival (PFS) and overall survival (OS). ABSTRACT.RESULTS: 201 patients were analysed for KRAS mutation (61.2% males; mean age 64.2 ± 8.6 years). KRAS mutations were identified in 36.3% of tumors (28.8% in codon 12, 7.4% in codon 13). The ORR in codon 13 patients compared to codon 12 and wild type patients was significantly lower (p = 0.008). There was a tendency for a better overall survival in KRAS wild type patients compared to mutants (p = 0.085). PFS in all patients was not different in the three KRAS genetic groups (p = 0.72). However, we found a marked difference in PFS between patients with codon 12 and 13 mutant tumors treated with infusional 5-FU versus capecitabine based regimens. ABSTRACT.CONCLUSIONS: Our data suggest that the type of KRAS mutation may be of clinical relevance under oxaliplatin combination chemotherapies without the addition of monoclonal antibodies in particular when overall response rates are important. ABSTRACT.TRIAL REGISTRATION NUMBER: 2002-04-017 BODY.BACKGROUND: The oncogene KRAS belongs to the protein family of small G-proteins and is mutated in 35-40% of colorectal cancers (CRC) [1]. RAS mutations are considered early events in colon carcinogenesis and are well conserved between primary tumor and corresponding metastases [2]. KRAS mutations tested routinely include six mutations in codon 12 and one mutation in codon 13. KRAS has been studied extensively as a prognostic marker in CRC, but results are still conflicting. Overall, there seems a tendency towards inferior outcome for patients with KRAS mutant tumors even in large randomized trials [3-7]. Importantly, KRAS has recently been identified as a strong predictive marker for patients with advanced CRC under anti-EGFR-treatment. Various studies demonstrated that while patients with tumors expressing wild type KRAS may benefit from anti-EGFR (epidermal growth factor receptor)-antibody treatment, patients carrying a mutated KRAS gene do not [5,8-10]. In contrast, two large trials using oxaliplatin-based chemotherapy-backbones did not confirm KRAS wild type to be a powerful predictor of treatment efficacy in metastatic CRC [4,11]. However, the studies published until recently did not differentiate between the different KRAS mutations and other histopathological or molecular features of their patients. The clinical observation, that some patients with KRAS mutation may respond to anti-EGFR-antibody therapy, as well as experimental data, that the biological effects of KRAS mutations may differ, was addressed recently by de Roock and Tejpar [12,13]. They reported that patients with codon 13 mutations in KRAS exhibit a worse overall prognosis with short overall survival times under standard chemotherapy, but may benefit from anti-EGFR-antibody therapy similar to wild type patients. These observations prompted us to look for the effect of the KRAS mutational status in correlation with response and survival data in patients with advanced colorectal cancer receiving oxaliplatin containing chemotherapy from a prospective randomized multicenter phase III trial of the German AIO study group. BODY.METHODS.PATIENTS: All patients participated in a prospective randomized phase III first-line palliative chemotherapy trial of advanced CRC of the AIO colorectal study group (Arbeitsgemeinschaft Internistische Onkologie of the German Cancer Society). The study was performed according to the Helsinki declaration and it was approved by the ethics review board of the Central Hospital Bremen and of the Medical Faculty of the Ruhr-University Bochum. The study design, patient characteristics, treatment plans and results of the clinical trial have been reported previously [14]. Briefly, a total of 474 patients were randomized to be treated with either 5-FU/folinic acid (FA) and oxaliplatin (FUFOX: oxaliplatin, 50 mg/m2; FA, 500 mg/m2; continuous 5-FU, 2,000 mg/m2/22 h; on day 1, 8, 15, 22; q day 36) or capecitabine and oxaliplatin (CAPOX: oxaliplatin, 70 mg/m2 on day 1 and 8; capecitabine, 2 × 1,000 mg/m2/day consecutively for 2 weeks, q day 22). No clinical factor was found to be predictive for definition of a subgroup of patients benefiting more or less from each fluoropyrimidine backbone. We here present data on a subcohort of 201 patients (42.4%) with available formalin-fixed paraffin-embedded tissue. Samples were retrieved from pathologists responsible for first diagnosis, pseudononymized and forwarded to the Institute of Pathology of the Ruhr-University Bochum, which was blinded to treatment allocation and prognostic outcomes. BODY.METHODS.DNA EXTRACTION AND MUTATION ANALYSIS: DNA was extracted from anonymized formalin-fixed paraffin embedded tissue samples. For each patient, five 10-μm sections were prepared. An additional representative 1-μm section was deparaffinized, stained with H&E, and analyzed for detailed morphology. Regions displaying tumor cellularity of >70% were marked and macrodissected. Tissue was extracted using QIAmp DNA Mini kit (Qiagen, Hilden, Germany). Real-Time PCR amplification for the most frequent seven KRAS mutations was performed using commercially available kits from DxS Ltd. (Manchester, UK) according to the manufacturer's instructions. This kit detects >95% of known KRAS mutations. Laboratory staff was blinded to the patient data and clinical outcome. BRAF mutation analysis to detect the V600E substitution was performed by RT-PCR (see supplemental file for further details). BODY.METHODS.STATISTICAL ANALYSIS: Chi-square test was used to evaluate the associations between KRAS status and other dichotomous variables. The analysis of progression-free survival (PFS) and overall survival (OS) was done using the Kaplan-Meier method and differences between subgroups were calculated by log-rank test. Data have been analyzed using SPSS 18.0 (Munich, Germany). All tests were two sided. For all tests, p values <0.05 were considered significant. BODY.RESULTS.PATIENT CHARACTERISTICS: The subcohort of patients successfully analyzed for KRAS mutations consisted of 201 patients (61.2% males with a mean age of 64.2 ± 8.6 years, range 35 – 82). Colon cancer was diagnosed in 131 (65.2%) patients while rectal cancer was diagnosed as primary tumor in 61 (30.3%) patients. Localization was not known for 9 cases (4.5%). 105 (52.2%) patients were treated with FUFOX, whereas 96 (47.7%) patients received CAPOX. 115 (57.2%) patients had synchronous metastases, whereas 59 (29.4%) had metachronous metastases. Whether metastases were synchronous or metachronous was not known for 27 ( 13.4%) patients (Table1). The subcohort reported here was representative of the complete study cohort with respect to age, gender, treatment plans (i.e. percentage of patients with FUFOX and CAPOX respectively). Median PFS and OS of the total ITT (intention-to-treat) population [14] and the biomarker subpopulation were fully comparable (PFS under FUFOX was 8.0 in the ITT cohort vs. 7.8 in the KRAS cohort; PFS under CAPOX was 7.1 vs. 7.0; OS under FUFOX was 18.8 in the ITT cohort vs. 17.5 in the KRAS subcohort and OS under CAPOX was 16.8 vs. 18.4). Table 1 Baseline characteristics of the investigated subcohort Biomarker population N % Age     Mean 64.15 years     Range 35–82 years     ≤60 y 58 28.9 >60 y 143 71.1 Sex     Female 78 38.8 Male 123 61.2 Treatment arm     FUFOX 105 52.2 CAPOX 96 47.8 Localization of primary tumor     Colon 131 65.2 Rectum 61 30.3 n.k. 9 4,5 T stage at initial diagnosis     T1 1 0.4 T2 15 7.5 T3 132 65.7 T4 49 24.4 n.k. 4 2.0 N stage at initial diagnosis     N0 46 22.9 N1 57 28.3 N2 89 44.3 n.k. 9 4.5 M stage at initial diagnosis     M0* 59 29.4 M1 115 57.2 n.k. 27 13.4 *M0 indicates metachronous metastatic disease at initial diagnosis of CRC, M1 indicates synchronous metastatic disease at initial diagnosis of CRC. n.k. = not known – there were no data available. BODY.RESULTS.MUTATION FREQUENCY: KRAS mutations could be successfully analyzed in 201 samples. We identified KRAS mutations in 73/201 (36.3%) tumors. 58 (28.9%) of KRAS mutations were located in codon 12, whereas 15 (7.46%) were found in codon 13(see Table2). The three most frequent KRAS alterations in our samples were c.35 G > A (G12D, n = 25; 12.4%), c.38 G > A (G13D, n = 15; 7.46%), and c.35 G > T (G12V, n = 13, 6.46%). In 128 patients no KRAS mutation was found (63.6%). In these patients we detected 13 mutations in the BRAF gene (V600E) (10% of wild type patients). Table 2   KRAS   mutation frequency in the investigated subcohort (   KRAS   Wild type: 128 (63.7%),   KRAS   Mutation 73 (36.3%))       relative frequency of occurrence (%) absolute frequency of occurrence CODON 12 mutation:       Aspartate (G12D) c.35G>A 12,4 25 Valine (G12V) c.35G>T 6,5 13 Alanine (G12A) c.35G>C 3,4 7 Cysteine (G12C) c.34G>T 3 6 Serine (G12S) c.34G>A 3 6 Arginine (G12R) c.34G>C 0,5 1 CODON 13 mutation:       Aspartate (G13D) c.38G>A 7,5 15 BODY.RESULTS.CORRELATION BETWEEN MUTATIONS AND RESPONSE RATE: Tumor response evaluation was available for 201 patients. Grouping all KRAS mutations together, mutated tumors were associated with a significantly lower response rate (RR; defined as partial or complete remission by RECIST) as compared to tumors without KRAS mutations (44.4% vs. 63.0%, p = 0.012). When patients with codon 13 mutated tumors were analysed separately the overall response rate in this cohort was 23% as compared to 49% in codon 12 mutated tumors and 63% in wild type tumors (Table3, p = 0.008). Disease control rates (DCR) were 77%, 81% and 88%, respectively, which was not statistically significant (Table3, p = 0.29). Table 3 Tumor response assessment and correlation to   KRAS   mutational status All patients All WT Codon 12 mutation Codon 13 mutation p-value No. of patients% 201 128 58 15   100 63.6 28.9 7.5 ORR% 55 63 49 23 0.008 95% CI (49–61) (54–71) (37–62) (8–51) (Chi-Square) DCR% 85 88 81 77 0.29 95% CI (80–90) (80–92) (68–89) (49–93) (Chi-Square) All patients. Percentages based on non-missing data, p-values for WT vs Codon 12 mutations vs codon 13 mutations; WT wild type; ORR overall response rate, DCR disease control rate. BODY.RESULTS.CORRELATION BETWEEN MUTATIONS AND PROGRESSION-FREE SURVIVAL: During follow-up, 170 of 201 evaluable patients had progressed. The median PFS in all patients of the KRAS subcohort was not statistically different in relation to the KRAS mutational status (wild type: 7.5 months, mutation codon 12: 8.2 months, mutation codon 13: 10.0 months; p = 0.71) (Figure1). However, when analysing the two treatment arms separately, we found a substantial, non-significant, difference in PFS in codon 13 mutated tumors versus codon 12 mutated tumors. While median PFS was as low as 6.1 months for codon 13 patients receiving infusional 5-FU, median PFS was 13.3 months in patients treated with capecitabine (HR: 2.52, p = 0.22). Patients with codon 12 mutations showed a trend towards the opposite effect: median PFS was 7.0 months under CAPOX therapy while median PFS was 9.9 months under FUFOX (HR: 0.62, p = 0.12) (Table4). Figure 1 Progression-free survival according to KRAS status. Table 4 PFS (progression free survival) according to   KRAS   mutation for the two different treatment arms PFS FUFOX CAPOX p-value (log rank)   n = 105 n = 96 HR (95% CI) All patients 8.2 months 6.4 months 0.18 0.81 (0.61-1.09) WT 8.1 months 6.4 months 0.29       0.82 (0.56-1.19) Codon 12 mutation 9.9 months 7.0 months 0.12       0.62 (0.33-1.14) Codon 13 mutation 6.1 months 13.3 months 0.22       2.52 (0.54-11.70) PFS, HR hazard ratio by cox regression, CI confidence interval, WT wild type. BODY.RESULTS.CORRELATION BETWEEN MUTATIONS AND OVERALL SURVIVAL: During follow-up 135 of 201 evaluated patients had died. We observed a trend towards a better survival time in patients with wild type tumors compared to those with a mutation of KRAS. The overall survival of wild type KRAS patients was 19.2 months, for patients with codon 12 mutations 15.6 months and for patients with codon 13 mutations 16.5 months. These differences were of marginal significance (p = 0.085) (Figure2). Figure 2 Overall survival according to KRAS status. Evaluating the different treatment arms separately, we found comparable survival times without significant differences (Table5). Since the survival curves seemed to separate for wild type KRAS and mutant KRAS patients at 14 months we hypothesised that this difference was most likely caused by the influence of post-study treatment. 71 out of 128 (55.4%) wild type patients received further lines of therapy while 32 out of 128 wild type patients (25%) received cetuximab. Out of 73 patients with KRAS mutations 42 were treated in further lines (57.5%) with 10 patients receiving cetuximab (13.7%). When analysing post progression survival (PPS) in these patients receiving second and further line therapies, patients receiving cetuximab had a significant better overall survival when compared to those patients under irinotecan therapy only, irrespective of the KRAS status. PPS in KRAS wild type patients: 32.2 months when cetuximab was given and 18.8 months when cetuximab was not included (p < 0.001). PPS for KRAS mutant patients: 27.9 months under cetuximab and 16.9 months without cetuximab (p = 0.032). Table 5 OS (overall survival) according to   KRAS   mutation for the two different treatment arms OS FUFOX CAPOX p-value (log rank)   n = 105 n = 96 HR (95% CI) WT 24.2 months 18.9 months 0.31       0.79 (0.51-1.24) Codon 12 15.6 months 15.5 months 0.54 mutation     0.83 (0.45-1.53) Codon 13 16.1 months 16.5 months 0.62 mutation     1.39 (0.37-5.37) OS, HR hazard ratio by cox regression, CI confidence interval. BODY.DISCUSSION AND CONCLUSIONS: We assessed the prognostic value of KRAS codon 12 and codon 13 mutations in tumor tissue from patients with advanced CRC recruited into a phase III clinical trial using CAPOX or FUFOX treatment regimens. This is the first randomized phase III trial retrospectively investigating the role of codon 13 mutations in advanced CRC patients treated with oxaliplatin combination chemotherapy only, without the addition of a monoclonal antibody. While the overall response rate in codon 13 patients was significantly lower, PFS was not different in the three KRAS mutational groups. Interestingly, we found a substantial difference in PFS between patients with codon 12 and 13 mutant tumors when looking at infusional 5-FU versus capecitabine based regimens. Patients with codon 13 mutations seem to benefit more in terms of PFS from the oral capecitabine based protocols. Moreover, there was a strong trend towards better overall survival in patients with wild type KRAS compared to all mutant KRAS patients. Lastly, when analysing OS in patients who received second and further line therapy we found that KRAS wild type and KRAS mutant patients alike showed a significantly higher OS post progression when treated with cetuximab. A number of studies have looked at the potential prognostic or predictive value of KRAS mutations on response rates and survival in patients with CRC and several studies found a negative impact of KRAS mutations on prognosis [3,7,15-17]. Recently, a number of randomized trials have included translational research programs to evaluate certain target genes and their role as prognostic or predictive markers in patients with metastatic disease. Thereby, the mutational status of KRAS has now been established as a strong predictive marker of resistance to anti-EGFR-antibody treatment [5,8,10], although some trials could not fully confirm these results [4,11]. We still do not exactly know whether KRAS mutations influence the response to other treatment regimens such as standard chemotherapy or bevacizumab combinations. While bevacizumab efficacy seems independent from the KRAS status [18], the activity of certain chemotherapeutic agents may be influenced by KRAS mutations. There, patients seem to do worse under oxaliplatin combinations when carrying a mutant KRAS gene within their primary cancer [4,5,7,16,19,20]. For example, in the recently reported COIN study patients under oxaliplatin/5-FU combinations showed a median PFS of 8.6 months in the wild type KRAS cohort while median PFS was only 6.9 months in the KRAS mutant cohort [4]. Similarily, ORR was lower in KRAS mutant patients receiving chemotherapy with oxaliplatin only (41% vs. 50%). Furthermore, another recent small study evaluated the KRAS status in 66 patients receiving a second line chemotherapy with oxaliplatin and infusional 5-FU refractory to 5-FU/irinotecan based chemotherapy [16]. This study found a significantly lower response rate (7% vs. 27%, p = 0.026) and significantly shorter median PFS (3.1 vs. 5.2 months, p = 0.007) for patients with mutant KRAS tumors compared to patients with wild type KRAS tumors under oxaliplatin containing therapy. Very recently, experimental and some clinical reports suggested that not all KRAS mutations behave alike [12,13,21]. In fact, there is evidence that patients carrying mutations in codon 13 of the KRAS oncogene which is found in about 8% of patients with advanced CRC have a substantially worse overall prognosis but may, on the other hand, benefit from anti-EGFR-treatment. In our study we found a similar rate of codon 13 mutations as described before. The overall response rate in patients with codon 13 mutations in our analysis was as low as 23%, significantly lower than in patients with wild type or codon 12 mutations. The CRYSTAL- and the OPUS-studies alike found low response rates in patients with codon 13 mutations treated with combination chemotherapy only (17% for irinotecan combinations and 33% for oxaliplatin combinations) [13]. In contrast to previous reports, ORR was substantially higher in this codon 13 mutant patient cohort when cetuximab was added. Our study was conducted using combination chemotherapy with oxaliplatin in first line treatment without the addition of monoclonal antibodies. Interestingly, the PFS and OS in our codon 13 cohort compared to the other mutated groups and other previously published works was rather long with a PFS of 10 months and an OS of 16.5 months [13]. We do not know why PFS and OS of the codon 13 cohort in our study was prolonged when response rates were as low as 23%. Either low patient numbers or a yet unknown functional mechanism of codon 13 mutated KRAS proteins within colon cancers may be responsible for the observation. The present analysis also suggests that there may be an interaction between the type of KRAS mutation and the mode of application of 5-FU, i.e. whether administered intravenously or orally as capecitabine. In particular, patients with codon 13 mutations showed longer median PFS intervals when receiving capecitabine compared to infusional 5-FU. Although the overall efficacy of infusional 5-FU and capecitabine in advanced CRC has been found to be comparable [22] there may be some patient subpopulations or treatment regimen where the mode of application of the fluoropyrimidine is more crucial. For example, anti-EGFR antibodies in wild type KRAS patients may only be active when infusional 5-FU regimens are used, but not when capecitabine based protocols are applied [4]. The potential resistance of KRAS mutated tumors to oxaliplatin containing regimens seems interesting and may reveal more general mechanisms of drug resistance in cancers. Oxaliplatin belongs to the platinum containing compounds like cisplatinum and carboplatinum. Metabolites of platinum compounds interact with DNA and form crosslinks. In addition, platinum-DNA-adducts strongly inhibit DNA polymerases and therefore act antineoplastic. Some authors have studied in cell culture systems and preclinical models the influence of oncogenic RAS mutations on the activity of platinum compounds and found that the nucleotide excision repair protein ERCC-1 may be upregulated through activated RAS. ERCC-1 may subsequently activate DNA repair capacity and thus mediate platinum resistance. A recent study evaluated the role of ERCC-1 mRNA levels in 191 patients treated with FOLFOX within the CONFIRM1 and CONFIRM2 studies. Low ERCC-1 gene expression was correlated with higher response rates to FOLFOX chemotherapy and better overall survival. In contrast patients with high ERCC-1 did not have benefit from FOLFOX chemotherapy [23]. Of note, we have examined the expression of ERCC-1 by immunohistochemistry in our cohort and found no obvious correlation with KRAS status, response rates or survival and ERCC-1 expression (data not shown). The analysis of the ERCC-1 expression levels by RT-PCR as reported by other studies has not been performed so far [24]. Median overall survival after first progression correlated with cetuximab-treatment in patients bearing KRAS wild type and mutant tumors alike. The role of codon 13 mutation in this setting appears minor since there were only two patients with a codon 13 mutated tumor in the cohort with KRAS mutations of whom we know about cetuximab application. However, there are at least two limitations to our analysis. First, numbers are low in particular in the cetuximab group and secondly addition of cetuximab was not randomized for. There seems to be a selection of patients with good performance status and good prognosis who received third line therapy compared to those who did not. Therefore, we can not draw definite conclusions from our analysis whether anti-EGFR antibodies are effective in patients with KRAS G13D mutations or not. We presently can not draw final conclusions regarding patient management from this study. It remains unclear whether chemotherapy backbones with irinotecan are less prone to interactions with mutated KRAS because data regarding this issue are conflicting. Independent validation of the findings is essential. Therefore, the standardized, thorough and comprehensive collection of tissue and blood samples of all trial patients within independent cancer tissue banks should be a major goal of modern clinical cancer trials. BODY.MISC: Anke Reinacher-Schick and Karsten Schulmann are contributed equally BODY.COMPETING INTERESTS: D. Arnold: Honoraria from Roche, Sanofi-Aventis. U. Graeven: Honoraria from Roche, Amgen, Sanofi-Aventis, Merck-Serono; Advisory Board Roche, Amgen. A. Reinacher-Schick: Honoraria from Amgen, Roche, Pfizer, Sanofi-Aventis; Advisory board member: Amgen, Roche, Pfizer; Studies sponsored by: Roche, Sanofi-Aventis. W. Schmiegel: Honoraria from Merck-Serono, Roche, Abott, Amgen, Astra-Zeneca, Pfizer, Falk; Advisory board member: Astra-Zeneca, Roche, Amgen; Studies sponsored by: Novartis, Amgen, Roche. K. Schulmann: Honoraria from Astra-Zeneca, Amgen, Falk; Travel support: Pfizer, Merck-Serono, Novartis. A. Tannapfel: Honoraria from Amgen, Roche, Pfizer, Sanofi-Aventis. Studies sponsored by: Roche, Sanofi-Aventis. The other authors declare that they have no competing interests. BODY.AUTHORS’ CONTRIBUTIONS: ARS and KS: analysis and interpretation of data, drafting of the manuscript, revising the manuscript critically, final approval of manuscript. DPM: interpretation of data, biostatistic analysis, revising the manuscript critically, final approval of manuscript. NB: acquisition of data, analysis and interpretation of data, final approval of manuscript. UG: conception and design, recruitment of patients, revising the manuscript critically, final approval of manuscript. MJ: acquisition of data, analysis and interpretation of data, final approval of manuscript. RG: recruitment of patients, revising the manuscript critically, final approval of manuscript. RP and WS: recruitment of patients, conception and design, revising the manuscript critically, final approval of manuscript. DA: conception and design, revising the manuscript critically, final approval of manuscript. AT: conception and design, data acquisition, analysis and interpretation of data, revising the manuscript critically, final approval of manuscript. All authors read and approved the final manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2407/12/349/prepub

TITLE: First-in-Human Evaluation of the Safety and Immunogenicity of an Intranasally Administered Replication-Competent Sendai Virus–Vectored HIV Type 1 Gag Vaccine: Induction of Potent T-Cell or Antibody Responses in Prime-Boost Regimens ABSTRACT: Background. We report the first-in-human safety and immunogenicity assessment of a prototype intranasally administered, replication-competent Sendai virus (SeV)–vectored, human immunodeficiency virus type 1 (HIV-1) vaccine. Methods. Sixty-five HIV-1–uninfected adults in Kenya, Rwanda, and the United Kingdom were assigned to receive 1 of 4 prime-boost regimens (administered at 0 and 4 months, respectively; ratio of vaccine to placebo recipients, 12:4): priming with a lower-dose SeV-Gag given intranasally, followed by boosting with an adenovirus 35–vectored vaccine encoding HIV-1 Gag, reverse transcriptase, integrase, and Nef (Ad35-GRIN) given intramuscularly (SLA); priming with a higher-dose SeV-Gag given intranasally, followed by boosting with Ad35-GRIN given intramuscularly (SHA); priming with Ad35-GRIN given intramuscularly, followed by boosting with a higher-dose SeV-Gag given intranasally (ASH); and priming and boosting with a higher-dose SeV-Gag given intranasally (SHSH). Results. All vaccine regimens were well tolerated. Gag-specific IFN-γ enzyme-linked immunospot–determined response rates and geometric mean responses were higher (96% and 248 spot-forming units, respectively) in groups primed with SeV-Gag and boosted with Ad35-GRIN (SLA and SHA) than those after a single dose of Ad35-GRIN (56% and 54 spot-forming units, respectively) or SeV-Gag (55% and 59 spot-forming units, respectively); responses persisted for ≥8 months after completion of the prime-boost regimen. Functional CD8+ T-cell responses with greater breadth, magnitude, and frequency in a viral inhibition assay were also seen in the SLA and SHA groups after Ad35-GRIN boost, compared with those who received either vaccine alone. SeV-Gag did not boost T-cell counts in the ASH group. In contrast, the highest Gag-specific antibody titers were seen in the ASH group. Mucosal antibody responses were sporadic. Conclusions. SeV-Gag primed functional, durable HIV-specific T-cell responses and boosted antibody responses. The prime-boost sequence appears to determine which arm of the immune response is stimulated. Clinical Trials Registration. NCT01705990. BODY: Despite significant progress in prevention and treatment of human immunodeficiency virus type 1 (HIV-1) infection, development of a safe and effective preventive HIV-1 vaccine remains a global priority [1, 2]. Several vaccine regimens have been tested for efficacy, but only one conferred modest, transient protection against HIV-1 acquisition [3]. Various methods have been evaluated to enhance responses to HIV vaccines, including booster injections, cytokine administration, adjuvants, electroporation, vector delivery systems, and prime-boost approaches [4–8]. Development of a vaccine that stimulates sustained humoral and/or cellular immunity at mucosal entry points may be critical for an HIV preventive vaccine. Although mucosally administered vaccines have been tested and licensed for other diseases [9–12], mucosal administration of an HIV preventive vaccine has seldom been evaluated [13]. Sendai virus (SeV) is a nonsegmented negative-sense RNA virus in the Paramyxoviridae family that can infect the upper respiratory tract [14–17]. As a live viral vector that is not pathogenic in humans, SeV offers several properties important for a successful vaccine: it does not integrate into the host genome, it replicates only in the cytoplasm without DNA intermediates or a nuclear phase, and it does not undergo genetic recombination. SeV is genetically and antigenically related to hPIV-1 [18–21]. A live nonrecombinant SeV vaccine against human parainfluenza virus type 1 (hPIV-1) administered intranasally in adults and young children was safe and immunogenic [22, 23]. SeV antibodies cross-reactive with hPIV-1 antibodies are present in most people [24]. Intranasal delivery of a vaccine could induce a first line of defense at mucosal points of entry and induce effective systemic immune responses [12, 25, 26]. Nonhuman primate studies with SeV bearing simian immunodeficiency virus (SIV) genes demonstrated protection against SIV challenge and evidence that SeV vectors may boost responses primed by other HIV-1 vaccines [27–29]. Intranasal administration and heterologous prime-boost administration were shown to reduce effects of preexisting immunity [29, 30]. In this study, we report the first-in-human safety and immunogenicity evaluation of a replication-competent SeV-vectored HIV-1 vaccine administered intranasally; the vaccine was administered intranasally at a lower dose (SL) or higher dose (SH) of SeV vector encoding clade A HIV-1 Gag (SeV-Gag), given alone or as a heterologous prime-boost with a nonreplicating adenovirus (Ad) serotype 35 HIV-1 vaccine containing genes HIV-1 encoding Gag, reverse transcriptase, integrase, and Nef (Ad35-GRIN) administered intramuscularly. The Ad35-GRIN was selected for these prime-boost regimens because it has well-known safety profile and robust immunogenicity in both US and African populations [4, 7, 8, 31]. BODY.METHODS.VOLUNTEERS AND STUDY DESIGN: This study was a multicenter, randomized, placebo-controlled, dose-escalation trial that was double blinded with respect to vaccine or placebo but not regimen. The doses were based on preclinical data [28, 29] and a nonrecombinant live SeV vaccine study in humans [23]; the initial group was administered a lower dose for safety. The study was conducted at Projet San Francisco (Kigali, Rwanda), the Kenya AIDS Vaccine Initiative Institute of Clinical Research (Nairobi, Kenya), and the St Stephen's AIDS Trust (London, United Kingdom). The objectives were to evaluate the safety and immunogenicity of 4 different 2-dose regimens (administered at 0 and 4 months) that comprised SeV-Gag administered at 2 × 107 (SL) or 2 × 108 (SH) cell infectious units and Ad35-GRIN vaccine administered at 1 × 1010 viral particles. Volunteers and clinical/laboratory personnel were blind to allocation between active vaccine and placebo. The participants were healthy HIV-negative adults 18–50 years of age engaging in behavior at low risk for HIV-1 infection; all women were nonpregnant and used an effective method of contraception until 4 months after the last vaccination (detailed inclusion/exclusion criteria are in Supplementary Materials). The respective local governmental ethics and regulatory bodies for each clinical research center approved the study. Written informed consent was obtained from each volunteer prior to undertaking any study procedure. The study was conducted in accordance with International Conference on Harmonization's good clinical practice and good clinical laboratory practice guidelines [32]. The study design is presented in Table 1 and in the Consolidated Standards of Reporting Trials diagram (Supplementary Figure 1). Volunteers in part I received low-dose SeV-Gag vaccine followed by Ad35-GRIN vaccine (SLA) or placebo. Following review of safety data from part I by an independent safety review board, a different set of volunteers was randomly assigned to participate in part II. Volunteers in part II received either the higher dose of SeV-Gag as a prime followed by Ad35-GRIN vaccine (SHA); an Ad35-GRIN prime given intramuscularly, followed by the higher-dose SeV-Gag boost given intranasally (ASH); prime-boost with the higher-dose SeV-Gag given intranasally (SHSH); or placebo. Table 1.Study Immunization Regimens and ScheduleGroupRegimenSubjects, No.Month 0Month 4Vaccine GroupPlacebo GroupVaccineRouteDoseaVaccineRouteDoseaASLA124SeV-GagIntranasal2 × 107Ad35-GRINIntramuscular1 × 1010BSHA124SeV-GagIntranasal2 × 108Ad35-GRINIntramuscular1 × 1010CASH125bAd35-GRINIntramuscular1 × 1010SeV-GagIntranasal2 × 108DSHSH124SeV-GagIntranasal2 × 108SeV-GagIntranasal2 × 108Abbreviations: Ad35-GRIN, adenovirus 35–vectored vaccine encoding Gag, reverse transcriptase, integrase, and Nef; ASH, Ad35-GRIN prime followed by SeV-Gag boost; HIV-1, human immunodeficiency virus type 1; SHA, higher-dose SeV-Gag prime and Ad35-GRIN boost; SHSH, higher-dose SeV-Gag prime and boost; SLA, lower-dose SeV-Gag prime and Ad35-GRIN boost; SeV-Gag, Sendai virus–vectored vaccine encoding HIV-1 Gag.a Data are 1 × 107 or 1 × 108 cell infectious units/100 μL per nostril (for SeV-Gag) or 1 × 1010 viral particles (for Ad35-GRIN).b Overenrollment was allowed per protocol; one additional volunteer, identified post unblinding as a placebo recipient, was enrolled. Each group had 16 volunteers: 12 vaccine recipients and 4 placebo recipients. Enrollment of an additional volunteer was allowed yielding a sample size of 65. Local and systemic reactogenicity were reported for days 0 through 14 following each vaccination, adverse events (AEs) were reported through month 1 following the second study vaccination, and serious adverse events (SAEs) were reported through the final study visit. Hematologic and biochemical parameters were assessed at 4 time points after vaccination (Supplementary Materials). Reactogenicity and AEs were assessed using an adapted version of the Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events, version 1.0. BODY.METHODS.STUDY VACCINES: The SeV-Gag vaccine is based on a replication-competent vector derived from the SeV Z strain [33] with HIV-1 subtype A gag inserted in the 3′ terminal region of the virus genome [34], upstream of the nucleoprotein gene. SeV-Gag vaccine and placebo were administered by syringe; the head was tilted back, and 100 μL was instilled into each nostril of the volunteer over approximately 3 minutes to allow absorption. The Ad35-GRIN vaccine is a recombinant, replication-defective Ad35 vaccine; it has been previously tested in 4 clinical trials [4, 7, 8, 31] and a recently completed trial in Kenya [35]. The Ad35-GRIN vaccine and placebo were both administered intramuscularly in 0.5 mL. The gag in SeV-Gag and Ad35-GRIN were fully homologous with regard to amino acid sequence. BODY.METHODS.LABORATORY ASSESSMENTS FOR SAFETY AND IMMUNOGENICITY: Hematologic and biochemical assays were conducted at the clinical sites in Africa and at a third-party accredited laboratory in the United Kingdom. Vaccine-induced seropositivity/seroreactivity was assessed in each country (Supplementary Materials). For detailed collection and immunogenicity testing methods, see the Supplementary Materials. Briefly, peripheral blood mononuclear cells (PBMCs) were processed and cryopreserved at each clinical site. Mucosal fluids from nasal swabs, parotid and transudated saliva, rectal secretions, and cervicovaginal secretions in females were processed as previously described [36, 37]. Colorectal biopsy specimens were pooled and disaggregated by collagenase digestion to isolate mucosal mononuclear cells within 6 hours of collection, and intracellular cytokine staining (ICS) assays were performed after an overnight rest as described elsewhere [38, 39]. T-cell responses were assessed by qualified interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) and ICS assays, using peptides matched to Gag, and by a functional viral inhibition assay, using a panel of 8 HIV-1 strains from subtypes A, B, C, and D [7, 8, 31, 40, 41]. An enzyme-linked immunosorbent assay (ELISA) was used with HIV-1 subtype B Gag p24 protein (BH10) to assess Gag p24 binding antibodies in serum and mucosal samples [7, 31, 36]. Serum neutralizing antibodies (NAbs) against SeV were assessed as described previously [24]. Samples for analysis of viral shedding were collected from the middle turbinate region, saliva, and urine on days 2, 5, 6, 7, and 9 (±1 day) after the first vaccination with SeV-Gag or placebo in the SLA, SHA, and SHSH groups as described in the Supplementary Materials. BODY.METHODS.STATISTICAL METHODS: The statistical methods are described in the Supplementary Materials. BODY.RESULTS.DEMOGRAPHIC CHARACTERISTICS AND PARTICIPANT FLOW AND RECRUITMENT: The study was conducted between March 2013 and March 2015. Of 65 volunteers, 36 (55.4%) were enrolled in Rwanda, 21 (32.3%) were enrolled in Kenya, and 8 (12.3%) were enrolled in the United Kingdom. Volunteers were first enrolled in group SLA only in Rwanda, followed by competitive enrollment at all sites for groups SHA, ASH, and SHSH. Twenty participants (30.8%) were female, and the mean age was 31.3 years (range, 19–48 years; Supplementary Table 1). All volunteers completed study vaccinations and visits per protocol (Supplementary Figure 1). BODY.RESULTS.VACCINE SAFETY AND TOLERABILITY: All vaccination regimens were generally well tolerated (Supplementary Figures 2A and 2B). There was no statistically significant difference in the frequency of grade 2 or higher upper or lower respiratory tract reactogenicity following any SeV-Gag vaccination, compared with placebo (Supplementary Table 2). All local reactogenicity events after Ad35-GRIN intramuscular injection were graded as mild or moderate. The frequency of grade 2 local pain, tenderness, erythema, and swelling following Ad35-GRIN vaccination was similar in the vaccine and placebo groups (Supplementary Table 3). Most systemic reactogenicity (chills, malaise, myalgia, headache, nausea, vomiting, and fever) was grade 1 or 2. The overall frequency of any grade 2 or higher systemic reactogenicity following any vaccination was similar in vaccine and placebo groups (Supplementary Tables 2 and 3). One volunteer (in the ASH group) reported grade 3 malaise on day 2 after Ad35 vaccination and grade 3 chills, malaise, and myalgia on day 0 after SeV-Gag vaccination (Supplementary Figure 2). There was no difference between groups in the proportion of volunteers with grade 2 or higher unsolicited AEs (P = .525; data not shown). The proportions of volunteers with any unsolicited respiratory AEs (cough, influenza-like illness, nasal congestion, pneumonia, and/or rhinitis) within 4 weeks of vaccination or at any time during the trial were not statistically significant between volunteers receiving SeV-Gag vaccination and placebo recipients (Supplementary Table 4). No vaccine-related SAE was reported, and no apparent pattern in clinical AEs or AEs determined by laboratory analysis was observed. No volunteers tested positive for vaccine-induced seropositivity/seroreactivity at the end of the study. Mucosal samples, including nasopharyngeal fluid, parotid gland saliva, oral fluid (transudate), and cervicovaginal and rectal secretions, were collected at 8 time points. Compliance was excellent for nasal and oral sampling, good for cervicovaginal sampling, but poor for rectal sampling (Supplementary Materials). BODY.RESULTS.SEV-GAG SHEDDING: Viral shedding samples were collected from the middle turbinate region, saliva, and urine on days 2, 5, 6, 7, and 9 after first vaccination with SeV-Gag or placebo. Overall, 20% of all samples (vaccine vs placebo, P = not significant) across all groups and visits (141 of 702) were positive by the cell infectious unit assay, which used immunostaining to detect cells infected with SeV. The polyclonal SeV antiserum used in this assay cross-reacts with hPIV-1, and a positive readout in this assay is either SeV or hPIV-1. Further analysis by PCR specific for SeV indicated that 12% of the cell infectious unit–positive samples (17 of 141) were positive for SeV and that all bore the intact HIV-1 gag insert. These 17 samples were from nasal swabs from 15 of 36 volunteers (42%) receiving only SeV-Gag. No SeV-Gag virus was detected in nasal samples after day 4 or at any time in saliva or urine (Supplementary Table 5). BODY.RESULTS.IFN-Γ ELISPOT FINDINGS: Few ELISPOT responses were detected after 1 or 2 vaccinations with SeV-Gag alone; in contrast, responses were detected in the heterologous regimens after the second vaccination (groups SLA, SHA, and ASH; Figure 1 and Table 2). Two weeks after the Ad35-GRIN boost, 12 of 12 volunteers in group SLA and 10 of 11 in group SHA demonstrated Gag-specific responses. Five group ASH volunteers had positive Gag responses at both time points, 1 group ASH volunteer had a positive Gag response after the first vaccination only, and 1 group ASH volunteer had a positive Gag response after the second vaccination only. The proportions were similar in groups SLA (100%) and SHA (90.9%; P = not significant). In the ASH group, 6 of 11 individuals (54.5%) had positive responses to the Gag peptide pool 2 weeks after the Ad35-GRIN prime (Table 2), and their responses remained steady after the SeV-Gag boost (Supplementary Table 6). In the SLA and SHA groups combined, the Gag response rate was significantly higher 2 weeks after the second vaccination (95.7%), compared with the rate in the ASH group 2 weeks after either the Ad35-GRIN prime or the SeV-Gag boost (54.5% for both; P = .008). Table 2.Human Immunodeficiency Virus Type 1 (HIV-1) Gag–Specific Interferon γ (IFN-γ) Enzyme-Linked Immunospot–Determined T-Cell Responses 2 Weeks After the First and Second VaccinationsGroup2 Weeks After First Vaccination2 Weeks After Second VaccinationSubjects, No.Response Rate,a Subjects, No. (%)Geometric Mean Responseb (95% CI)Range of Positive ResponsesbSubjects, No.Response Rate,a Subjects, No. (%)Geometric Mean Responseb (95% CI)Range of Positive ResponsesbSLA100 (0)3 (2–5)...1212 (100)275 (138–546)53–1740SHA110 (0)2 (1–5)...1110 (91)222 (101–488)60–1061SA210 (0)3 (2–4)...2322 (96)248 (154–400)53–1740ASH116 (55)54 (25–120)48–381116 (55)59 (30–113)43–485SHSH120 (0)3 (2–7)...120 (0)4 (2–8)...Placebo170 (0)2 (1–3)...160 (0)2 (1–4)...Abbreviations: Ad35-GRIN, adenovirus 35–vectored vaccine encoding HIV-1 Gag, reverse transcriptase, integrase, and Nef; ASH, Ad35-GRIN prime followed by SeV-Gag boost; CI, confidence interval; HIV-1, human immunodeficiency virus type 1; SA, SHA and SLA groups combined; SHA, higher-dose SeV-Gag prime and Ad35-GRIN boost; SHSH, higher-dose SeV-Gag prime and boost; SLA, lower-dose SeV-Gag prime and Ad35-GRIN boost; SeV-Gag, Sendai virus–vectored vaccine encoding HIV-1 Gag.a Excludes samples in which the prevaccination values for the Gag peptide pool were positive (ie, cross-reactive).b Data are IFN-γ spot-forming units per million peripheral blood mononuclear cells. Figure 1.Administration of a Sendai virus (SeV)–vectored vaccine encoding human immunodeficiency virus 1 (HIV-1) Gag (SeV-Gag) primes Gag-specific T-cell responses detected by interferon γ enzyme-linked immunospot analysis. The y-axis shows spot-forming units (SFU)/106 peripheral blood mononuclear cells (PBMCs) on a log scale. All responses reflect subtraction of background spots. Black circles denote response below the cutoff, defined in the Materials and Methods, to the Gag peptide pool; red circles denote response above the cutoff to the Gag peptide pool. The overlaid box plots summarize the responses (ie, median value, the 1st and 3rd quartiles, and the 5th and 95th percentiles). Red bars represent median values. The placebo responses are combined for all groups. The prime-boost regimens as follows: priming with a lower-dose SeV-Gag given intranasally, followed by boosting with an adenovirus 35–vectored vaccine encoding HIV-1 Gag, reverse transcriptase, integrase, and Nef (Ad35-GRIN) given intramuscularly (SLA); priming with a higher-dose SeV-Gag given intranasally, followed by boosting with Ad35-GRIN given intramuscularly (SHA); priming with Ad35-GRIN given intramuscularly, followed by boosting with a higher-dose SeV-Gag given intranasally (ASH); and priming and boosting with a higher-dose SeV-Gag given intranasally (SHSH). Arrows show the timing of vaccinations in each group. In the x-axis, weeks represent the time after the most recent vaccination, to facilitate cross-regimen comparisons. With respect to the magnitude of the response, the IFN-γ ELISPOT responses to Gag 2 weeks after Ad35-GRIN receipt were greater after SeV-Gag priming (in groups SLA and SHA combined) than after the Ad35-GRIN prime (in the ASH group), with geometric mean responses of 248 and 54 IFN-γ spot-forming units (SFU)/106 PBMCs (P = .002; Table 2). The geometric mean responses in the SLA and SHA groups 2 weeks after Ad35-GRIN receipt were similar (275 and 222 IFN-γ SFU/106 PBMCs, respectively; P = .73). The response rate and magnitude in group SLA waned over time but remained constant in group SHA; the ASH group had lower but more-stable response rates (Figure 1 and Supplementary Table 6). BODY.RESULTS.ICS FINDINGS: Flow cytometry was used to characterize the phenotype and polyfunctionality of the response. Low responses were detected in group ASH 2 weeks after the Ad35-GRIN prime and 2 weeks after the SeV-Gag boost (Figure 2 and Supplementary Table 7). However, in groups SLA and SHA, 2 weeks after receipt of the Ad35-GRIN boost, the median magnitude of the Gag-specific CD4+ T-cells expressing IFN-γ, IL-2, or TNF-α was significantly higher than after receipt of each dose of the ASH regimen (P = .006 and P = .013, respectively). After prime and boost (in the SLA group), the median number of Gag-specific CD4+ T-cells expressing IFN-γ, IL-2, or TNF-α increased approximately 45-fold, from 0.005% to 0.225% (Figure 2A and Supplementary Table 7). In contrast, 2 weeks after receipt of the Ad35-GRIN boost in the SLA and SHA groups and 2 weeks after receipt of each dose of the ASH regimen, no significant difference was observed in the magnitude of the Gag-specific CD8+ T-cells (Figure 2B and Supplementary Table 7). Figure 2.Characterization of human immunodeficiency virus (HIV-1) Gag–specific CD4+ and CD8+ T-cell responses. Gag-specific CD4+ and CD8+ T-cell responses as assessed by intracellular cytokine staining (ICS; A and B). Time points displayed are 2 weeks after the indicated vaccination. T-cell responses were evaluated by 7-color ICS to assess the expression of interleukin 2 (IL-2), interferon γ (IFN-γ), and tumor necrosis factor α (TNF-α) with peptides matched to the Gag peptide pool. The percentage of T cells expressing at least 1 cytokine (IL-2, IFN-γ, or TNF-α) is shown. Boxes represent interquartile ranges (IQRs), and whiskers extend to the 5th and 95th percentiles. Red bars represent median values. The placebo responses are combined for all groups. For the polyfunctional responses (bottom panels C and D), each dot represents a single volunteer. The prime-boost regimens as follows: priming with a lower-dose SeV-Gag given intranasally, followed by boosting with an adenovirus 35–vectored vaccine encoding HIV-1 Gag, reverse transcriptase, integrase, and Nef (Ad35-GRIN) given intramuscularly (SLA; red dots); priming with a higher-dose SeV-Gag given intranasally, followed by boosting with Ad35-GRIN given intramuscularly (SHA; green dots); priming with Ad35-GRIN given intramuscularly, followed by boosting with a higher-dose SeV-Gag given intranasally (ASH; orange dots); and priming and boosting with a higher-dose SeV-Gag given intranasally. Ad35-GRIN prime alone is represented by blue dots, and placebo is represented by purple dots. Gag-specific CD4+ polyfunctional T-cell responses predominated over CD8+ T-cell responses in groups SLA and SHA, with the majority of volunteers positive for all 3 cytokines tested (Figure 2C). Gag CD8+ T-cell responses were highest in group SHA, with the majority being either triple positive or double positive for IFN-γ and TNF-α (Figure 2D). Fresh colorectal biopsy mucosal mononuclear cells and PBMCs were tested in parallel by ICS in 16 volunteers, including placebo recipients who consented to the procedures. The mucosal mononuclear cells and PBMC samples had responses to SEB and/or CMV as expected, but only 1 vaccinee, in group SHA, had Gag-specific responses in both PBMCs and mucosal mononuclear cells 2 weeks after prime-boost (data not shown). BODY.RESULTS.VIRAL INHIBITION ASSAY: Viral inhibition was detected in 2 of 9 and 3 of 9 individuals after SeV-Gag prime and in 11 of 12 and 9 of 9 individuals after the Ad35-GRIN boost in groups SLA and SHA, respectively (P = not significant). When the vaccination order was reversed, in the ASH group, viral inhibition was detected after the Ad35-GRIN prime in 9 of 10 volunteers but in only 4 of 10 after the SeV-Gag boost (Table 3 and Supplementary Figure 3A and 3B). The viral inhibition response rate after the boost in groups SLA and SHA combined was greater than in the ASH group (20 of 21 [95%] vs 4 of 10 [40%]; P = .0017, by the Fisher exact 2-tailed test). Table 3.Viral Inhibition Responses 2 Weeks After the First and Second VaccinationsVariableSLASHASAASHPlacebobPrimeBoostPrimeBoostPrimeBoostPrimeBoostAnyPositive results, proportiona2/911/123/99/95/1820/219/104/101/4Log10 p24 inhibition Median0.881.720.952.070.891.851.290.850.79 Maximum2.124.862.134.832.134.863.142.771.76Breadthc Median0.06.00.04.00.06.03.00.00.0 Mean0.224.750.674.780.475.003.001.200.50 Range0–10–70–41–80–40–80–60–60–2Abbreviations: Ad35-GRIN, adenovirus 35–vectored vaccine encoding HIV-1 Gag, reverse transcriptase, integrase, and Nef; ASH, Ad35-GRIN prime followed by SeV-Gag boost; HIV-1, human immunodeficiency virus type 1; SA, SHA and SLA groups combined; SHA, higher-dose SeV-Gag prime and Ad35-GRIN boost; SLA, lower-dose SeV-Gag prime and Ad35-GRIN boost; SeV-Gag, Sendai virus–vectored vaccine encoding HIV-1 Gag.a Data are no. positive/no. tested. Positive results correspond to any positive value per individual over all 8 virus isolates. Any virus isolate that was positive at baseline (which occurred in 3 individuals) was counted as negative. All other values are calculated using all results over all virus isolates per volunteer.b At least 1 positive result after prime or boost. Other statistics include both prime and boost values.c Breadth corresponds to the no. of positive virus isolates per individual. In the SLA and SHA groups, the median log inhibition after the SeV-Gag prime was 0.88 and 0.95, respectively, and it increased significantly to 1.72 and 2.07 after the Ad35-GRIN boost (P < .0001). In group ASH, the median log inhibition after receipt of Ad35-GRIN alone was 1.29, decreasing to 0.85 after SeV-Gag receipt (Table 3). The magnitude of viral inhibition after boost in the SLA and SHA groups combined (1.85) was greater than after the Ad35-GRIN prime (1.29) and the SeV-Gag boost (0.85) in group ASH (P < .0001 for each comparison). A response to 2 viruses was detected after the second dose of placebo in 1 volunteer. The breadth of viral inhibition was assessed by the number of viruses inhibited out of a panel of 8 viruses from multiple HIV-1 clades. The greatest median breadth was observed in groups SLA and SHA after the Ad35-GRIN boost (6 and 4, respectively), compared with a median breadth of 3 after the Ad35-GRIN prime alone in the ASH group (Figure 3 and Table 3). Viral inhibition was not assessed in group SHSH. Figure 3.Sendai virus (SeV)–vectored vaccine encoding human immunodeficiency virus 1 (HIV-1) Gag (SeV-Gag) enhances the breadth of inhibition of a panel of diverse HIV-1 isolates. The breadth of inhibition among 8 viruses was assessed at baseline (bl) and specified time points 2 weeks after the indicated vaccination (vac). Lines represent median values, whiskers represent the 1st and 3rd quartiles, and gray dots represent individual responses. The placebo (Pbo) responses are combined for all groups. The prime-boost regimens as follows: priming with a lower-dose SeV-Gag given intranasally, followed by boosting with an adenovirus 35–vectored vaccine encoding HIV-1 Gag, reverse transcriptase, integrase, and Nef (Ad35-GRIN) given intramuscularly (SLA); priming with a higher-dose SeV-Gag given intranasally, followed by boosting with Ad35-GRIN given intramuscularly (SHA); priming with Ad35-GRIN given intramuscularly, followed by boosting with a higher-dose SeV-Gag given intranasally (ASH). BODY.RESULTS.GAG P24 ANTIBODIES: Gag p24–specific IgG and IgA were measured in serum samples at baseline, 2 and 16 weeks after the first vaccination, and 2, 16, 32, and 48 weeks after the second vaccination. In SLA, SHA, and SHSH recipients, GMTs were negative at all time points (ie, they were ≤100) after prime and boost. The GMTs were significantly higher (P < .0001, by the Kruskal-Wallis test) in the ASH group after the SeV boost than in the other groups (53, 53, 245, 50, and 59 in the SLA, SHA, ASH, SHSH, and placebo groups, respectively; Figure 4). Two volunteers in group ASH were excluded from this analysis owing to positive titers at baseline. ASH titers decreased to a GMT of 72 at 48 weeks after SeV-Gag receipt. Gag p24–specific IgA responses were detected sporadically and at low titers (data not shown). Figure 4.Human immunodeficiency virus type 1 (HIV-1) Gag p24 immunoglobulin G (IgG) geometric mean titers (GMTs) after the following prime-boost combinations: priming with a lower-dose SeV-Gag given intranasally, followed by boosting with an adenovirus 35–vectored vaccine encoding HIV-1 Gag, reverse transcriptase, integrase, and Nef (Ad35-GRIN) given intramuscularly (SLA); priming with a higher-dose SeV-Gag given intranasally, followed by boosting with Ad35-GRIN given intramuscularly (SHA); priming with Ad35-GRIN given intramuscularly, followed by boosting with a higher-dose SeV-Gag given intranasally (ASH); and priming and boosting with a higher-dose SeV-Gag given intranasally (SHSH). Titers below the level of detection (ie, <100) were given a value of 50. Arrows indicate vaccination time points for each group. Placebo responses are combined for all groups. Abbreviation: CI, confidence interval. BODY.RESULTS.SEV NABS: Most volunteers from all groups had a preexisting positive titer of serum SeV NAbs, with similar magnitudes and rates in vaccine and placebo recipients before and after vaccination (Supplementary Figure 4). There was no correlation between SeV NAb titers at baseline and the magnitude of Gag ELISPOT responses 4 weeks after Ad35-GRIN receipt in groups SLA and SHA and Gag-specific ELISA responses 2 weeks after the SeV boost in group ASH (Supplementary Figure 5). Mucosal secretions were not tested for SeV NAbs. BODY.DISCUSSION: This was the first-in-human trial assessing safety and immunogenicity of a replication-competent SeV-vectored HIV-1 vaccine; it was administered in prime-boost regimens with Ad35-GRIN in healthy volunteers. SeV has been safely tested as a nonrecombinant Jennerian vaccine against hPIV-1 in adult and pediatric populations [22, 23]. Both SeV-vectored and Ad35-vectored vaccines were well tolerated, and adverse events were not significantly different from those in placebo recipients. Mucosal sampling was well accepted in this study when it was limited to saliva, nasal, or cervicovaginal fluids, but rectal fluid collection and biopsy had more-limited acceptance, as in previous studies [36, 37]. We postulated that delivery of SeV-Gag by the intranasal route might induce mucosal immune responses and circumvent preexisting immunity to SeV, allowing immune responses to the vaccine insert, as shown previously in animal models [30, 42]. Despite preexisting SeV NAbs in all groups and a lack of persistent SeV shedding indicative of replication, there was a clear take of SeV-Gag, as indicated by much greater Gag-specific T-cell and antibody responses in the heterologous regimens, compared with either vaccine given once. Mucosal antibody responses were weak and sporadic, and neither mucosal application nor parenteral priming or boosting amplified mucosal responses. Remarkably, the vaccines induced very different immune responses when given in a different order. As a homologous regimen, 2 doses of SeV-Gag induced minimal humoral and cellular immune responses. In contrast, SeV-Gag primed T-cell responses for a subsequent boost by Ad35-GRIN, while SeV-Gag boosted humoral responses after priming with Ad35-GRIN. The strongest Gag-specific T-cell responses were detected by ELISPOT and ICS assays after the SeV-Gag prime and Ad35-GRIN boost, with no clear effect of the SeV-Gag dose. Functional viral inhibition responses mediated by T cells [41] with greater breadth, magnitude, and frequency were also seen in groups SLA and SHA after the Ad35-GRIN boost. The frequency and magnitude of Gag ELISPOT responses in the SLA and SHA groups combined were equivalent to those of Ad35-GRIN given twice intramuscularly, indicating that SeV-Gag given intranasally provides as strong a prime as an Ad vector given intramuscularly [31]. A so-called hidden prime has been postulated previously in studies of DNA vaccines followed by Ad vector boosts [43, 44], in which plasmid DNA vaccines with or without electroporation and/or molecular adjuvants such as interleukin 12 or interleukin 15 elicit very modest T-cell and antibody responses in humans, but prime for anamnestic responses when Ad vectors are given as a boost [8, 45–49]. One dose of SeV-Gag appears to be equivalent to 3 doses of DNA vaccine (up to 8 mg) in terms of priming responses and is perhaps more effective at priming than a highly attenuated VSV-Gag delivered intramuscularly [6, 50, 51]. The ICS studies demonstrated that SeV-Gag stimulated CD4+ T cells, which may have provided help for the development of CD8+ T cells [44]. It was not possible in this study to determine how SeV-Gag provides this potent T-cell priming; its ability to infect mucosal cells after intranasal delivery may be important. A direct comparison of 2 routes of delivery could help to resolve this issue. In contrast, Gag-specific antibody responses were detected only when Ad35-GRIN was given first and boosted by SeV-Gag in group ASH. Previous studies have shown low-to-negligible Gag-specific antibody responses after administration of a single dose of Ad35-GRIN [31]. After priming with Ad35-GRIN and boosting with SeV-Gag, the Gag ELISA responses were equivalent to those seen with Ad35-GRIN given twice intramuscularly, indicating that SeV-Gag given intranasally provides as strong a boost as an Ad vector given intramuscularly [31]. In summary, intranasal delivery of SeV is feasible, safe, and immunogenic in the presence of preexisting systemic antivector antibodies; neither antibody nor T-cell responses were equivalent at the 2 SeV-Gag doses tested. The type of response elicited was determined by the order of vaccines in the heterologous regimen, as SeV-Gag priming mainly induced cellular immunity whereas SeV-Gag boosting mainly induced serum humoral immune responses against Gag. Mucosal antibody responses were weak and sporadic, and only 1 participant had a mucosal T-cell response. Whether mucosal antibodies are necessary or sufficient for protection against sexual transmission of HIV is unknown; if they are important, a more powerful immunogen or different regimen will be needed. Further studies to elucidate the mechanism of this antibody–T cell shift may be warranted. These data suggest that intranasal delivery of a viral vector capable of limited replication as part of a heterologous prime-boost regimen may be a valuable way to stimulate immune responses, even in the presence of preexisting antivector antibodies. SeV-Gag was shown to prime for T-cell responses and to boost antibody responses, but in this configuration the SeV-Gag by itself is not sufficiently immunogenic for further development. BODY.STUDY GROUP MEMBERS: The S001 Study Team includes the following individuals: Rosine Ingabire, Gina Ouattara, Alan Steele, Anne Gumbe, Kundai Chinyenze, Sabrina Welsh, Carl Verlinde, Deborah King, Cynthia Bishop, Paramesh Chetty, Lorna Clark, Mumtaz Booley, Devika Zachariah, Kristen Syvertsen, Kamaal Anas, Marloes Naarding, Emmanuel Cormier, Jim Ackland, and Mamoru Hasegawa. BODY.SUPPLEMENTARY DATA: Supplementary materials are available at http://jid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author. Supplementary Data

TITLE: The Effect of Stretching Exercises on Severity of Restless Legs Syndrome in Patients on Hemodialysis ABSTRACT.BACKGROUND: The restless legs syndrome is a sensorimotor disorder that is very common in patients on hemodialysis. Due to pharmacological treatments which have their own side effects, nowadays, studies have turned to non-pharmacological treatments. ABSTRACT.OBJECTIVES: The present study aims to assess the effect of stretching exercises on the severity of restless legs syndrome in patients on hemodialysis. ABSTRACT.PATIENTS AND METHODS: This clinical trial study was conducted on 33 patients who had been identified using diagnostic criteria from the hemodialysis ward of Hasheminejad Hospital in Tehran. Participants were randomly divided into the intervention group (n = 17) and control group (n = 16). Stretching exercises were performed on legs during the dialysis for half an hour, three times a week for 8 weeks in intervention group. Data were collected by using the international restless legs syndrome study group scale. ABSTRACT.RESULTS: The results showed that the majority of participants were suffering from moderate restless legs syndrome. The symptom severity of this syndrome meaningfully changed eight weeks after intervention in the intervention group compared to the control group (P < 0.001). ABSTRACT.CONCLUSIONS: The results highlighted the significance of training and performing the stretching exercises during dialysis for the purpose of improving restless legs syndrome symptoms and the quality of care of hemodialysis patients. BODY.1. BACKGROUND: Restless legs syndrome (RLS) is a neurological movement disorder that affects a large number of people (1). RLS prevalence studies show that 10% of the adult population is affected by this disorder (2). The outbreak of the disease is more common in end stage renal disease (ESRD) patients than general population (3). 20% to 70% of the dialysis patients report symptoms of RLS (4). Some studies also indicate that 33% of ESRD patients suffer from RLS (3). Restless legs syndrome has been shown to have a significant impact on the quality of life of the patients on hemodialysis mainly because of the poor quality of sleep and inadequate rest (2). RLS is a disorder that gives the patient unpleasant sensations in his limbs, especially legs. These sensations make an urge to move the legs to stop the sensations (5). The syndrome is a sensorimotor disorder that is characterized by restlessness symptoms that lead to an irresistible urge to move some body parts (2). This comes in the evening after sitting for a long time and the sensations are expressed in tickling, pain, itching, and stretching feelings. The symptoms of RLS may appear at any age; 12% of RLS symptoms appear before ten (6). But overall, RLS often appears in middle age (5). Many factors of RLS are unknown or hereditary (7), but it is usually most common in people suffering from chronic renal diseases, diabetes, iron deficiency anemia, Parkinson's, neuropathy, pregnancy, use of caffeine, calcium channel blocker drugs, lithium, and sedative drugs withdrawal (5). The special criteria for diagnosis of RLS have been introduced by the international restless legs syndrome study group (IRLSSG) and including; 1) a uncontrolled urge to frequently move the limbs due to RLS symptoms, 2) temporary relief of the unpleasant symptoms by moving; 3) worsening of the symptoms after resting or lack of movement, 4) worsening of the symptoms in the evening or at night (8). Worsening of the symptoms in night in patients with moderate to severe RLS leads to sleep disorder. As a result, RLS patients may experience anxiety and sleep deprivation or drowsiness during the day that can disrupt the daily functioning of the patient (9). Also RLS causes sleep disorder, daily fatigue, depression, disruption of the ability to work and social isolation (10). These problems lead to poor quality of life and have adverse effects on social activities and family life (9). Regarding this information, studies have shown that the reduced quality of life in hemodialysis patients with RLS is possible because of inadequate sleep quality and quantity (2). Common medications for RLS has been dopamine agonists or benzodiazepines, but the drugs have some side-effects (1). Initially, to treat RLS, non-pharm logical treatments like sleep hygiene, warm bath, massages, reduction of stimulating factors such as caffeine and alcohol should be tried (11). Recently, some researcher has been conducted on the effect of changing lifestyle like performing exercises (1). Since the symptoms of the syndrome appear or worsen in resting and immobility times, these symptoms may improve by moving. Therefore, moderate exercise can be helpful (2). Exercises like walking, massaging, stretching, swimming, and stationary cycle can help relieve symptoms (12). The effects of exercise on RLS symptoms are unknown. A research by Ohayon et al. (13) in 2002 showed that severe physical activities like high intensity sports just before bedtime exacerbate the symptoms of RLS. Also Aukerman and Sakkas concluded that a daily mild exercise program can alleviate the RLS symptoms (1, 2). BODY.2. OBJECTIVES: Review of recent studies showed that we need more research about the effects of exercise on RLS symptoms. Therefore, the present study aims to assess the effects of stretching exercises on severity of RLS symptoms in patients on hemodialysis because stretching exercises are easily applicable by the patients and are applied on legs and don't cause disruption in dialysis. BODY.3. PATIENTS AND METHODS.3.1. DESIGN AND SAMPLE: This study was a randomized controlled trial conducted on hemodialysis patients from the hemodialysis ward of Hasheminejad Hospital affiliated to Tehran University of Medical Sciences in Tehran. Given the study by Aukerman et al. (1) with a confidence interval of 95% and testing power of 90%, the sample size was calculated 13 for each group, but totally 18 people were considered for each group considering the possible dropout, so 36 people were enrolled. BODY.3. PATIENTS AND METHODS.3.2. INCLUSION AND EXCLUSION CRITERIA: The inclusion criteria were having diagnostic criteria for RLS according to IRLSSG, age over 18, at least 6 months since the start of dialysis treatment, being on the weekly list of dialysis for 3 times a week and each time for 3 to 4 hours, having no mental or physical disability, being completely alert, having acceptable hearing and speaking ability for answering the questions, absence of any infection, injuries, and peripheral neuropathy or vascular problems in legs, absence of orthopedic problems, and confirmation of RLS by physician in research centers. The exclusion criterion was refusal to do the stretching exercises for 3 consecutive sessions and overall 6 sessions. BODY.3. PATIENTS AND METHODS.3.3. PROCEDURE AND INSTRUMENTS: To identify the patients with RLS, initially, four diagnostic criteria were given to all the patients at the hemodialysis ward. Patients, who answered "yes" to all 4 questions, were examined by the physician for confirmation of RLS. The patients with RLS were included in the study consensually and randomly divided into intervention and control groups. Initially, the stretching exercises were taught to the intervention group during two sessions. After the training, the intervention group participated in 8 weeks of a stretching exercise program three times a week during the last two hours of hemodialysis session and each time for 30 minutes. The researcher closely monitored the exercises. Each session involved 5 minutes of initial warm-up. Then, the participants did stretching exercises on legs for 20 minutes and then cooled down for 5 minutes. The stretching exercises that were confirmed by a sports medicine specialist were applied to the legs and included hip rotation to the sides, quadriceps stretch, knee to chest stretch, hamstring stretch, gluteal stretch, straight leg raise, side lying leg lift and so on. Each muscle was stretched to three sets of ten. Duration of each stretching was 5 seconds. After training sessions these exercises was performed by the participants under the supervision of the researcher. For assessment of the effect of stretching exercises on severity of RLS we used the international RLS study group (IRLSSG) Scale. This scale was completed at baseline, at the end of the 4th week, and at the end of the 8th week by the subjects. The demographic information was collected by a questionnaire. The scale for assessing the severity of RLS involved 10 items each having 5 options. Every question had 0 to 4 points. The maximum score was 40 and a high score indicated the severity of disease. The participants with less than 10 points were mild, 11 to 20 points were moderate; 21 to 30 points were severe and above 31 was very severe. All interviewers and personnel who handled the questionnaires were blinded to study allocation of the participants. In order to determine content validity and reliability of the IRLSSG questionnaire, Cornbrash's alpha coefficient was used in this study. The validity of IRLSSG questionnaire was 0.87. The Data were collected in two months in 2012 and were analyzed using SPSS16 software program and Fisher's statistical test, two-sample Kolmogorov-Smirnov test, t-test and ANOVA. A P value < 0.05 was considered statistically significant. The study was introduced and approved by the Ethics Committee of the Tehran University of Medical Sciences in 09 September 2012. All participants signed written consent after full explanation of the procedure. BODY.4. RESULTS: After screening all the patients who were in the list of hemodialysis ward and identifying patients with RLS, 36 people were included in the study. They were divided randomly into two 18-member control and intervention groups. One patient was excluded from the intervention group because of not completing stretching exercises sessions, and two patients were excluded from the control group (one of them had died and the other one was not cooperative). In total, the study continued with 33 people (17 patients in the intervention group and 16 patients in the control group). According to the results, 51.5% of the participants were men and 30.3% were above 66-years-old. Most participants were married (63.6%) and jobless or retired (78.1%). The most common diseases among the participants were hypertension (48.5%) and diabetes (42.4%) and the most used drug was amlodipine (45.5%). The results also showed that 100% of the participants were unaware that they were suffering from RLS and they did not take any medications to relieve symptoms of RLS. An overwhelming majority of the participants (87.9%) did not do exercises and any sports regularly. Most of the participants were suffering from medium to severe of RLS and there was no meaningful difference between the intervention and control groups in the beginning of the study. Repeated ANOVA test showed that the severity of RLS scores (according to IRLSSG scale) decreased meaningfully in the intervention group by 8 weeks, but these scores didn't change significantly in the same period in the control group (P < 0.001) (Table 1). Table 1. Comparison of Severity of RLS Scores in Control and Intervention Group at Baseline, End of 4 th Week and End of 8 th Week by ANOVA Test a , b , c Variable Baseline (A) End of 4 th Week (B) End of 8 th Week (C) ANOVA Test Control group d 19 ± 4.78 18.43 ± 4.47 19.06 ± 4.71 0.55 Intervention group e 18.94 ± 5.44 17.47 ± 5.1 12.41 ± 3.79 < 0.001 a Total severity of RLS scores range from zero to 40 with higher values being indicative of severe RLS symptoms. b P < 0.05 are considered significant. c Values are expressed as Mean ± SD. d A and B, NS (P = 0.262); A and C, NS (P = 0.937); B and C, NS (P = 0.307). e A and B, SN (P = 0.001); A and C, SN (P < 0.001); B and C, SN (P < 0.001). Also the results of t-test show that the changes in the severity of RLS symptoms before intervention and after 4 weeks were not statistically meaningful between intervention and control groups. However, the changes were meaningful at the end of the 8th week (P < 0.001) (Table 2). Table 2 . Comparison of Severity of RLS Scores in Control and Intervention Group at Baseline, End of 4 th Week and End of 8 th Week by t-test a , b , c Variable Intervention Group d Control Group e t-test Value P Value Baseline (A) 18.94 ± 5.44 19 ± 4.78 -0.33 0.97 End of 4 th week (B) 17.47 ± 5.1 18.43 ± 4.47 -0.57 0.56 End of 8 th week (C) 12.4 1± 3.79 19.06 ± 4.71 -4.48 < 0.001 a Total severity of RLS scores range from zero to 40 with higher values being indicative of severe RLS symptoms. b P < 0.05 are considered significant. c Values are expressed as Mean ± SD. d A and B, SN (P = 0.001); A and C, SN (P < 0.001); B and C, SN (P < 0.001). e A and B, NS (P = 0.262); A and C, NS (P = 0.937); B and C, NS (P = 0.307). BODY.5. DISCUSSION: In this study, the severity scores in the intervention group were 18.94 in the beginning and it decreased to 12.41 at the end of the 8th week. The changes are totally significant, but not in the control group. The present research indicated that stretching exercises for 8 weeks (24 sessions) on legs during hemodialysis improves the RLS symptoms (Figure 1). Figure 1.Comparison of RLS Severity Scores Changes in Intervention and Control Groups During the Eight Week's InterventionNo changes in the RLS severity scores were found in the Control Group while statistically significant differences were found only between week 0 and week 8 for the intervention group (P < 0.05). Since the hemodialysis patients are often disabled and they spend most of their time on dialysis beds, they are usually inactive. Therefore, the proposed stretching exercises can be very helpful because they focus only on lower body limbs and have no side effects such as disruption in the hemodialysis treatment due. The results of this research are compatible with the other studies. Sakkas et al. (2) showed that aerobic exercises (as a mild sport) during hemodialysis reduce the severity of RLS symptoms in 16 weeks. Aukerman et al. (1) showed that meaningful changes in the severity of RLS symptoms are observed from onset of first week to the end of 6th week. However, in this study no meaningful change was detected after the 6th week to the end of 12th week. Therefore, 8 weeks exercise can suffice to prove the effectiveness of stretching exercises on the severity of RLS symptoms. In contrast, some researchers like Ohayon et al. (13) showed that relatively severe physical activity like high intensity sports before bedtime is meaningfully linked to the severity of RLS symptoms. Therefore, hemodialysis patients are recommended to avoid severe and hard sport programs especially before bedtime. According to this fact, the present study examined the effects of intradialytic mild or moderate stretching exercises. The present research also showed that most participants (78.51%) were jobless or retired that leads to inactivity. Since the RLS symptoms appear or worsen in immobility, physical activity improves the symptoms (13). Therefore, having a mild, regular exercise program can help to alleviate the RLS symptoms and will enhance the health of the people suffering from the syndrome. Scientists believe despite the fact that the real cause of RLS is still unknown; the syndrome is more common among diabetic patients (5). On the other hand, diabetes is one of causes of renal failure; it is more common among hemodialysis patients. This study proved this finding too. Because in our findings, the most common diseases in patients suffering from RLS were diabetes. Regarding the positive effect of stretching exercises on hemodialysis patients, nurses in charge of diabetic patients are also recommended to train them in the exercises to improve their life quality. This study has found that the most common drug used by the participants is amlodipine. This drug blocks calcium channels and is used for heart diseases. According to different studies, people who use calcium channel blockers are more vulnerable to RLS (5). It seems that there is a meaningful relation between this drug and RLS. It is recommended that further studies are needed to assess this relationship. An absolute majority of the participants in this study did not perform any exercises regularly. Although hard exercises, particularly before sleep can worsen the RLS symptoms and may lead to sleep disorder and chronic insomnia, moderate or light exercises program can be helpful during the day to improve RLS symptoms. This study showed that regular stretching exercises on legs during hemodialysis can help alleviate the severity of RLS symptoms. The exercises have no side effects and no cost and can be done without help. Nurses can help alleviate the RLS symptoms by training the patients and thus take a positive step towards healthcare objectives.

TITLE: Effects of High-Intensity Interval Training versus Continuous Training on Physical Fitness, Cardiovascular Function and Quality of Life in Heart Failure Patients ABSTRACT.INTRODUCTION: Physical fitness is an important prognostic factor in heart failure (HF). To improve fitness, different types of exercise have been explored, with recent focus on high-intensity interval training (HIT). We comprehensively compared effects of HIT versus continuous training (CT) in HF patients NYHA II-III on physical fitness, cardiovascular function and structure, and quality of life, and hypothesize that HIT leads to superior improvements compared to CT. ABSTRACT.METHODS: Twenty HF patients (male:female 19:1, 64±8 yrs, ejection fraction 38±6%) were allocated to 12-weeks of HIT (10*1-minute at 90% maximal workload—alternated by 2.5 minutes at 30% maximal workload) or CT (30 minutes at 60–75% of maximal workload). Before and after intervention, we examined physical fitness (incremental cycling test), cardiac function and structure (echocardiography), vascular function and structure (ultrasound) and quality of life (SF-36, Minnesota living with HF questionnaire (MLHFQ)). ABSTRACT.RESULTS: Training improved maximal workload, peak oxygen uptake (VO2peak) related to the predicted VO2peak, oxygen uptake at the anaerobic threshold, and maximal oxygen pulse (all P<0.05), whilst no differences were present between HIT and CT (N.S.). We found no major changes in resting cardiovascular function and structure. SF-36 physical function score improved after training (P<0.05), whilst SF-36 total score and MLHFQ did not change after training (N.S.). ABSTRACT.CONCLUSION: Training induced significant improvements in parameters of physical fitness, although no evidence for superiority of HIT over CT was demonstrated. No major effect of training was found on cardiovascular structure and function or quality of life in HF patients NYHA II-III. ABSTRACT.TRIAL REGISTRATION: Nederlands Trial Register NTR3671 BODY.INTRODUCTION: Heart failure (HF) is a common disease with an increasing prevalence worldwide [1], and is characterized by a low 5-year survival of 35–55% [2–4]. Several studies have indicated that physical fitness is an important prognostic factor for HF patients, in which a low physical fitness is associated with higher mortality rates [5,6]. Previous studies demonstrate that exercise training can improve physical fitness [7], cardiac function [8], vascular function [9,10], and quality of life [7] in HF patients [11]. Therefore, exercise training is recommended for HF patients and encompasses a central component of cardiac rehabilitation [12]. Most previous studies examining the impact of exercise training in HF patients have adopted exercise at moderate-intensity. Recent studies explored the impact of high-intensity interval training (HIT), which can be described as short periods of exercise performed at high-intensity (>80–85% peak oxygen uptake [13–15]), alternated by periods of active or passive rest. Wisløff et al. suggested a superior effect of HIT compared to continuous training (CT) on physical fitness, cardiovascular function and quality of life in HF patients [16]. This study applied an intensive HIT training regimen in HF patients and found large improvements in fitness levels. After this promising study, Fu et al. found similar results [17], but not all subsequent studies [18–22], reinforced the superior effect of HIT compared to CT in HF patients. Previous studies that revealed a superior effect of HIT adopted relatively long bouts of high-intensity exercise (3–4 min) [16,17], followed by active 'rest' periods of 3 minutes at an intensity up to 70% of maximal heart rate. Performance of such long bouts of exercise at high-intensity and/or 'rest' periods at such vigorous intensity levels may not be feasible for all HF patients. Furthermore, most previous training studies have applied a training frequency of three times per week, which is time consuming. Whilst training frequencies of twice per week can be sufficient to induce a positive effect on fitness levels [23], no previous study examined the efficacy of HIT with lower training frequency or whether there is a difference between such HIT and CT training protocols. Therefore, we explored the benefits of a feasible HIT-protocol with high-intensity bouts of moderate duration (i.e. 1 min), the active rest at relatively low intensity (i.e. 30% maximal workload), and a training frequency of twice a week. In this study, we examined whether 12-weeks of CT or HIT is effective and feasible for HF patients, and whether this HIT-protocol leads to superior effects on fitness, cardiovascular function and quality of life compared to CT. We hypothesize that the HIT-protocol is feasible, whilst the effects on physical fitness, cardiovascular function and quality of life in HF patients New York Heart Association (NYHA) class II-III are superior compared to CT. BODY.METHODS.SUBJECTS: We included 29 patients (65±8 yrs) diagnosed with HF classified as NYHA class II-III, with a history of left ventricular ejection fraction (LVEF) ≤45% (assessed by 2D/4D echocardiography). Patients were recruited from the department of Cardiology of the Radboud university medical center and the Canisius-Wilhelmina Hospital (Nijmegen, the Netherlands) and through advertisements. Patients with HF due to congenital heart disease or HF caused by valve pathology were excluded. Other exclusion criteria were: diabetes mellitus (type 1 or 2), hypercholesterolemia (total cholesterol >6.5mmol/L), severe renal failure (glomerular filtration rate<30 mL/min/1.73m2), exercise-induced ischemia (i.e. ECG abnormalities suggestive for ischemia on maximal exercise testing), severe co-morbidities (e.g. COPD GOLD ≥3), pathology that restricts patients from participation to exercise (e.g. orthopedic/neurological disorders interfering with movement), pre-menopausal women or women on hormone replacement therapy, and subjects with contra-indications for maximal exercise testing [24]. Subjects had to be in a clinically and pharmacologically stable situation (>3 months) prior to participation. The Medical Ethical Committee of the Radboud university medical center approved this study (CMO region Arnhem-Nijmegen) on October 26th 2010. This trial is registered in the Dutch Trial Register (NTR3671) 3 months after the start due to practical reasons. The authors confirm that all ongoing and related trials for this intervention are registered. Written informed consent was obtained from each subject before participation in this study. Subject recruitment was done between July 2011 and September 2014. Follow-up lasted until February 2015. This study was monitored by a data safety monitoring board, which also approved the submission of this study. BODY.METHODS.EXPERIMENTAL PROTOCOL: Subjects reported to our laboratory for a medical screening, after which they were allocated to 12-weeks moderate-intensity CT or HIT. In addition, to assess changes over time in HF patients, we included a control group consisting of HF patients who were unable to participate in the training program due to geographical reasons or time-constraints, which was tested before and after a 12-week control period. Before and after the 12-week intervention/control period, subjects underwent a maximal incremental cycling test to determine physical fitness, echocardiography to examine cardiac function and structure, and vascular ultrasound measurements to examine peripheral artery vascular function and structure. Finally, questionnaires were used before and after the intervention to assess health-related quality of life and HF symptoms. All measurements were performed in the Radboud university medical center (Nijmegen, the Netherlands). Due to the nature of the study design and practical reasons, blinding participants and researchers was not possible. BODY.METHODS.MEASUREMENTS.SUBJECT CHARACTERISTICS: We determined height, weight (Seca 888 Scale, Seca, Hamburg, Germany), body mass index, body fat percentage [25], and waist and hip circumference. Furthermore, we obtained heart rate and blood pressure (manually, WelchAllyn, Maxi-Stabil 3, NY, USA), an electrocardiogram to determine heart rhythm, and a venous blood sample to determine fasted glucose and (total) cholesterol concentrations. BODY.METHODS.MEASUREMENTS.PRIMARY OUTCOME—PHYSICAL FITNESS: An incremental maximal cycling test was performed on a cycle ergometer (Ergoline, Ergoselect 200k, Bitz, Germany). Subjects were instructed to pedal (>60rpm) whilst workload was increased 10–15 Watt/min, depending on the expected physical fitness of the participant (based on sex, age, height, and previous results on exercise testing). During exercise, breath-by-breath gas analysis was recorded continuously (LabManager V5.32.0). For the termination of maximal exercise testing we adhered to recent guidelines [24]. Peak oxygen uptake (VO2peak) was defined as the highest oxygen uptake (30-second average). BODY.METHODS.MEASUREMENTS.SECONDARY OUTCOME—PHYSICAL FITNESS: Oxygen uptake at the anaerobic threshold (AT) was determined using the V-slope method [26]. Peak oxygen uptake and oxygen uptake at the AT was also expressed as a percentage of the predicted maximal oxygen consumption [27]. Ventilatory efficiency was defined as the slope of the ventilation to the carbon dioxide production (VE/VCO2 slope) calculated over the linear phase of the response up to the AT. The maximal oxygen pulse (oxygen consumption per heart rate, O2/HR) was determined (10-second average). The presence of chronotropic incompetence, defined as a heart rate reserve <80% of predicted [28], was noted. Prior to testing, all medication was continued. BODY.METHODS.MEASUREMENTS.SECONDARY OUTCOME—VASCULAR FUNCTION AND STRUCTURE: Subjects were prepared according to guidelines for the assessment of flow-mediated dilation (FMD) [29]. Subjects were instructed to continue medication, but to refrain from diuretics the day of testing for practical reasons. The measurements were performed in a temperature-controlled room (22.5±0.7°C). Prior to testing, subjects rested in the supine position for 10 minutes. Vascular function measurements were performed using a 10-MHz multifrequency linear array probe attached to a high-resolution ultrasound machine (Terason T3000, Burlington, MA, USA). We examined brachial and superficial femoral artery endothelial function using the FMD according to the guidelines [29]. Subsequently, we measured the brachial artery maximal diameter and blood flow responses to ischemic handgrip exercise, as described in previous studies [30,31]. The peak blood flow provides a valid and accepted index of resistance artery size and remodelling and the brachial artery diameter response for maximal dilating capacity [30]. We examined carotid artery intima-media thickness (IMT), which represents a surrogate measure for atherosclerosis [32]. Finally, we examined the endothelium-independent dilation of the brachial artery by examining the diameter response to an exogenous nitric oxide donor (sublingual administration of 400μg glyceryl trinitrate (GTN)). BODY.METHODS.MEASUREMENTS.SECONDARY OUTCOME—CARDIAC FUNCTION: Transthoracic (4D) echocardiography was performed with an ultrasound scanner (Vivid E9, General Electric Healthcare, Horten, Norway) with M5-S and V4 probe according to the guidelines of the American Society of Echocardiography [33]. Echocardiographic images were analyzed post-hoc with EchoPAC software (version 112, General Electric Healthcare, Horten, Norway). From 4D-images we assessed: left ventricular end-diastolic volume, left ventricular end-systolic volume, stroke volume, LVEF, cardiac output, and cardiac index. Left ventricular longitudinal, circumferential, radial, and area strain were analyzed by 4D speckle tracking. Moreover, we measured the isovolumetric contraction and relaxation time from tissue Doppler tracings of the lateral and septal mitral annulus. To describe diastolic function we obtained the following parameters by (tissue) pulsed-wave Doppler tracings: peak mitral flow velocity during early (E) and late (A) diastole, the systolic (S) and diastolic (D) inflow velocity over the pulmonary valve, and the peak mitral annulus velocity during early filling (E') of the lateral and septal mitral annulus. The E/A-ratio, S/D-ratio, and E/E'-ratio were calculated. BODY.METHODS.MEASUREMENTS.SECONDARY OUTCOME–QUESTIONNAIRES: To measure health-related quality of life, the SF-36 Health Survey was used [34,35]. Additionally, we used the Minnesota living with Heart Failure Questionnaire (MLHFQ), to measure patient perceptions of the effects of HF on their physical, psychological and socioeconomic lives [36]. BODY.METHODS.12-WEEK INTERVENTION: Training was performed twice a week for 12 weeks in a rehabilitation setting or hospital and was supervised by physiotherapists. When a participant missed a training session, this session was rescheduled to ensure a total of 24 training sessions (i.e. 100% compliance). Training was performed on a cycle ergometer (Lode Corival, Procare, Groningen, The Netherlands). Both the CT- and HIT-session started with a warm-up of 10-minutes at 40% of maximal workload (Watt) as obtained from the cardiopulmonary maximal exercise test at baseline, and concluded with a cooling-down of 5-minutes at 30% of maximal workload. Workload was increased during the 12-week training period based on the Borg scores of perceived exertion, to maintain a sufficient training stimulus when physical fitness was expected to improve. BODY.METHODS.12-WEEK INTERVENTION.CT-GROUP: CT consisted of 30-minutes at 60–75% of maximal workload. Training intensity was controlled using the Borg score (scale 6–20) [37], aiming at a Borg score of 12–14 during the training session, as recommended in the current exercise guidelines for HF patients [12]. Borg score and heart rate were determined after the warm-up, at 20, 30 and 40 minutes of exercise, and after the cooling-down. BODY.METHODS.12-WEEK INTERVENTION.HIT-GROUP: HIT consisted of 10 periods of 3.5-minutes of exercise, consisting of intervals of 1-minute at 90% of maximal workload, and 2.5-minutes at 30% of maximal workload, aiming at a Borg score of 15–17 during the high-intensity intervals. Borg score and heart rate were determined at the end of the warm-up, after repetition 1, 3, 7 and 10, and after the cooling-down. BODY.METHODS.12-WEEK INTERVENTION.CONTROL GROUP: Control subjects were instructed not to alter their daily physical activities. BODY.METHODS.STATISTICAL ANALYSIS: We have made a pre-study sample size calculation based on previous studies examining the difference in effect between CT and HIT. Some studies suggest n = 2–3 per group is sufficient [16,17], whilst data from others suggest several thousand subjects must be recruited to detect differences between CT and HIT [19]. We rationalized that n = 10–20 will provide (clinically) meaningful insight into the effect of CT versus HIT. Therefore, using a conservative approach (accounting for drop-outs), we aimed for n = 20 for both exercise training groups (and n = 10 in the control group). Data was analyzed using IBM SPSS Statistics 20.0 (IBM Corp., Armonk, NY, USA). Parameters were checked for normality using a Kolmogorov-Smirnov test. When data was not normally distributed, a non-parametric alternative was used or natural logarithmic data transformation was applied. Categorical and nominal parameters were compared with a Chi-Square test. Baseline characteristics of the groups were compared with a 1-way ANOVA or Kruskal-Wallis test when data was not normally distributed. A 2-way repeated measures ANOVA was used to examine the impact of exercise training (time-effect), and whether the change differs between HIT and CT (time*group-effect). When a significant main effect (time) or interaction-effect (time*group) was observed, post-hoc tests with least-significant difference were used to identify differences between and within groups. When data for this 2-way comparison was not normally distributed, we used individual tests to examine the effect of time, group and time*group. Changes in the control group were tested with a paired Student's t-test, or Wilcoxon test when data was not normally distributed. To control for the potential impact of baseline diameter on FMD [38], we used logarithmically transformed diameter data and adopted a univariate General Linear Model with baseline arterial diameter as a covariate, to compare differences between groups. Potential drop-outs were left out of the analysis and were not replaced. A Pearson correlation coefficient was determined for the relation between baseline quality of life and exercise-induced changes in quality of life. Data are presented as mean±standard deviation (SD), unless stated otherwise. Significance level was set at P<0.05. BODY.RESULTS: For this study, 59 HF patients were screened. Fifteen patients did not meet inclusion criteria (screen failures) and 11 patients declined to participate after screening due to lack of time (n = 10) or change in health (n = 1) (Fig 1). We allocated 24 patients to HIT or CT. Nine HF patients were included in the control group (non-randomized). In both the HIT- and CT-group, 2 drop-outs were reported after allocation (71±2yrs; male:female 3:1; NYHA class II:III 3:1), which makes the total drop-out 17%. A patient in both training groups dropped out due to musculoskeletal complaints and a patient in both training groups dropped out due to progression of HF. Twenty patients in the training-groups and nine controls completed the study and were available for final analysis. The groups were not significantly different in age, body mass index, NYHA class, etiology of HF, blood pressure, heart rate, LVEF and physical fitness (Table 1). The control group consisted of significantly more females than the CT-group (P = 0.028, Table 1). Cardiovascular medication use is documented in Table 1. 10.1371/journal.pone.0141256.g001Fig 1Flow-chart of the inclusion of subjects. 10.1371/journal.pone.0141256.t001 Table 1 Subject characteristics and cardiovascular medication. CT (n = 10) HIT (n = 10) Control (n = 9) Age (yrs) 64±8 63±8 67±7 0.57 Sex (male:female) 10:0 * 9:1 5:4 0.028 Body weight (kg) 89.7±11.9 87.6±23.6 77.0±10.5 0.16 Height (cm) 177±5 177±3 174±9 0.66 Body mass index (kg/m 2 ) 28.9±4.7 28.1±7.5 25.4±2.7 0.24 NYHA class (II:III) 8:2 8:2 8:1 0.84 Etiology (Isch:Non-isch) 8:2 7:3 5:4 0.51 Systolic blood pressure (mmHg) 132±23 132±18 130±25 0.98 Diastolic blood pressure (mmHg) 83±11 79±10 78±14 0.48 Resting heart rate (/min) 59±11 57±7 60±10 0.80 Maximal heart rate (/min) † 129±19 126±16 120±15 0.53 Chronotropic incompetence (yes:no) † 5:4 8:2 6:1 0.33 VO 2peak † (mL/min/kg) 21.0±3.4 19.1±4.1 17.4±5.8 0.26 VO 2peak † (% of predicted VO 2peak ) 86±8 79±17 81±22 0.63 LVEF (%) 38±6 37±6 40±11 0.84 Medication     Angiotensin converting enzyme-inhibitors 5 (50%) 6 (60%) 8 (89%) 0.19     Angiotensin II receptor antagonists 4 (40%) 4 (40%) 1 (11%) 0.30     Aldosterone antagonist 6 (60%) 7 (70%) 8 (89%) 0.36     Diuretics (loopdiuretics) 7 (70%) 6 (60%) 4 (44%) 0.50     β-blockers 10 (100%) 9 (90%) 9 (100%) 0.37     Antiplatelet drugs 6 (60%) 4 (40%) 3 (33%) 0.47     Coumarin derivates 4 (40%) 7 (70%) 4 (44%) 0.35     Statins 10 (100%) 9 (90%) 4 (44%) § 0.007 Data is presented as mean±SD. P-values refer to a 1-way ANOVA. † Data was unavailable for 1 subject in the CT-group and 3 subjects in the control-group. * Significantly less females compared to the control-group. § Lower compared to CT-group and HIT-group. BODY.RESULTS.EXERCISE TRAINING: When averaging all training sessions, CT was performed at 66±5% of maximal workload, whilst the high-intensity intervals during HIT were performed at 102±7% of maximal workload (P<0.001). CT was performed at 81±7% of maximal heart rate, and the high-intensity intervals during HIT were performed at 83±9% of maximal heart rate (P = 0.70). Borg-scores during CT and HIT were 13±1 and 14±1 respectively (P = 0.27). BODY.RESULTS.IMPACT OF EXERCISE TRAINING.PHYSICAL FITNESS: VO2peak tended to increase after training (P = 0.06), whilst VO2peak/kg did not change after exercise (P = 0.10). A significant increase after training was found in the VO2peak related to predicted VO2peak (%), maximal workload, oxygen uptake at the AT and maximal oxygen pulse (Table 2). No significant differences were observed between both interventions (Table 2). 10.1371/journal.pone.0141256.t002 Table 2 Maximal incremental cycling test. CT (n = 10) HIT (n = 10) P-value Pre Post Pre Post Time Group Time*Group VO 2peak (mL/min) 1881±214 1887±27 1662±562 1792±559 0.06 0.44 0.08 VO 2peak (mL/min/kg) 21.2±3.6 21.3±3.7 19.1±4.1 20.4±4.3 0.10 0.14 0.09 # VO 2peak (% pred. VO 2peak ) 86±8 87±10 79±17 85±16 0.044 0.48 0.08 Max. workload (Watt) 145±22 152±26 126±38 142±45 <0.001 0.24 0.07 Max. heart rate (/min) 129±19 132±24 126±16 125±15 0.78 0.30 0.51 VE/VCO 2 slope 32.2±3.3 32.7±5.8 28.7±5.8 29.4±7.7 0.52 0.18 0.91 VO 2 at AT (mL) 1030±287 1248±388 1033±319 1090±225 0.041 0.54 0.22 Max. O 2 /HR (mL) 16.2±2.2 16.7±2.8 14.0±4.0 15.4±3.8 0.006 0.25 0.13 Data is presented as mean±SD. P-values refer to a 2-way repeated measures ANOVA between the two training groups. One subject in the CT-group did not reach VO 2peak , and therefore only VE/VCO 2 slope and VO 2 at AT could be determined. # For statistical reasons, data was analyzed with three separate tests to determine time , group and time*group P-values. BODY.RESULTS.IMPACT OF EXERCISE TRAINING.VASCULAR FUNCTION/STRUCTURE: We found no significant changes in brachial and superficial femoral artery diameter, peak blood flow, and FMD (Table 3). No change in endothelium-independent dilation of the brachial artery was observed after training for both groups (Table 3). Furthermore, we found no significant impact of HIT or CT on carotid artery IMT or IMT-to-lumen ratio (Table 3). 10.1371/journal.pone.0141256.t003 Table 3 Brachial (BA) and superficial femoral artery (SFA) endothelium-dependent vasodilation through flow-mediated dilation (FMD), peak diameter and endothelium-independent dilation (GTN), and common carotid artery (CCA) intima-media thickness (IMT). CT (n = 10) HIT (n = 10) P-value Pre Post Pre Post Time Group Time*Group BA diameter (mm) 4.5±0.5 4.5±0.5 4.4±0.9 4.4±0.8 0.83 0.68 0.86 BA FMD (%) 5.2±2.5 4.8±3.0 5.3±2.6 4.7±2.5 0.33 0.92 0.98 BA FMD (%, scaled) 5.3±2.5 4.8±2.5 5.2±2.5 4.6±2.5 0.47 >0.999 0.91 # BA SR AUC (s, 10 3 ) 19.9±9.6 18.6±7.4 17.8±9.2 22.3±7.7 0.42 0.80 0.14 BA GTN (%) 17.6±7.0 16.3±6.5 16.8±8.7 15.2±4.9 0.33 0.74 0.92 BA GTN (%, scaled) 17.9±4.5 16.6±4.5 16.1±4.5 14.6±4.5 0.42 0.27 0.94 BA FMD-GTN ratio 0.34±0.21 0.33±0.24 0.42±0.30 0.34±0.15 0.46 0.63 0.82 BA CADC (%) 10.6±6.4 11.3±4.8 16.1±7.4 13.4±5.5 0.52 0.12 0.26 BA CADC (%, scaled) 11.0±4.2 12.0±4.2 15.0±4.2 12.8±4.2 0.72 0.11 0.27 BA peak blood flow (mL/min) 794±139 862±261 711±264 556±165 0.38 0.56 0.46 SFA diameter (mm) 7.1±1.2 6.9±1.2 6.7±1.1 6.5±1.0 0.38 0.48 0.88 SFA FMD (%) 3.3±1.9 5.1±4.8 4.2±1.8 3.4±2.8 0.58 0.74 0.19 SFA FMD (%, scaled) 3.5±2.9 5.1±2.9 4.1±2.9 3.1±2.9 0.74 0.50 0.19 SFA SR AUC (s, 10 3 ) 9.2±5.6 10.4±10.8 13.5±7.2 6.9±3.9 0.11 0.80 0.07 CA IMT (mm) 0.80±0.13 0.76±0.20 0.72±0.12 0.67±0.16 0.20 0.24 0.96 CA IMT-to-lumen ratio 0.12±0.02 0.11±0.02 0.11±0.02 0.11±0.02 0.40 0.59 0.45 Data is presented as mean±SD. P-values refer to 2-way repeated measures ANOVA between the 2 training groups. # For statistical reasons, data was analyzed with three separate tests to determine time , group and time*group P-values. Due to technical problems, BA GTN/FMD-GTN ratio/peak blood flow was available for 9 subjects in the HIT-group and SFA FMD was available for 8 subjects in the HIT-group. CCA IMT and IMT-to-lumen ratio were available for 8 subjects in each group. SR AUC ; shear rate area-under-the-curve. CADC; conduit artery dilating capacity. BODY.RESULTS.IMPACT OF EXERCISE TRAINING.CARDIAC FUNCTION/STRUCTURE: Most of the parameters of cardiac systolic function, left ventricle strain, or diastolic function demonstrated no change after HIT or CT (Table 4). Negligible but significant changes were found in area strain and isovolumetric contraction time (Table 4). 10.1371/journal.pone.0141256.t004 Table 4 Echocardiographic left ventricular volumes, systolic function, strain and diastolic function. CT (n = 10) HIT (n = 10) P-value Systolic function Pre Post Pre Post Time Group Time*Group LVEDV (ml) 154±24 159±28 194±39 204±44 0.26 0.002 0.64 # LVESV (ml) 98±14 102±19 134±32 132±40 0.87 0.037 0.63 Stroke volume (ml) 56±13 57±13 61±14 72±16 0.06 0.16 0.12 LVEF (%) 36±4 36±5 32±7 36±9 0.09 0.57 0.07 Cardiac output (L/min) 3.5±0.6 3.4±0.7 3.5±0.8 4.3±1.0 0.20 0.21 0.07 Cardiac index (L/min/m 2 ) 1.7±0.3 1.6±0.3 1.7±0.4 2.1±0.5 0.22 0.14 0.08 Longitudinal strain (%) -9±3 -9±3 -9±3 -8±3 0.60 0.47 0.38 # Circumferential strain (%) -10±2 -10±3 -9±3 -8±3 0.22 0.43 0.19 Radial strain (%) 23±7 22±6 23±8 20±8 0.13 0.71 0.48 Area strain (%) -17±4 -15±6 -17±5 -14±5 0.044 0.73 0.97 IVCT-L (ms) 52±7 50±10 49±12 58±7 0.18 0.56 0.029 IVCT-S (ms) 57±14 59±11 53±9 56±11 0.35 0.46 0.87 Diastolic function IVRT-L (ms) 145±32 149±27 142±27 159±27 0.13 0.75 0.38 IVRT-S (ms) 160±36 148±22 164±41 170±37 0.60 0.45 0.22 # E/A 1.15±0.71 1.17±0.89 1.53±1.42 1.60±1.53 0.49 0.36 0.59 # S/D 1.38±0.74 1.17±0.34 1.00±0.40 1.26±0.59 0.85 0.48 0.14 E/E’-L 6.8±1.9 6.7±1.9 10.3±4.4 9.8±6.3 0.71 0.07 0.74 E/E’-S 10.1±4.1 11.1±5.2 12.6±9.8 11.8±11.2 0.93 0.67 0.42 Data is presented as mean±SD. P-values refers to 2-way repeated measures ANOVA between the 2 training groups. # For statistical reasons, data was analyzed with three separate tests were performed to determine time , group and time*group P-values. 4D data was available for 7 patients in the CT-group and 8 patients in the HIT-group. IVCT-l, IVRT-C, IVRT-S and E/E’-L was available for 9 patients in the HIT-group. IVCT-S and E/E’-S was available for 8 patients in the HIT-group. IVCT-L and S/D ratio was available for 9 subjects in the CT-group. IVRT-L and E/A ratio was available for 8 subjects in the CT-group. LVEDV; left ventricular end-diastolic volume. LVESV; left-ventricular end-systolic volume. IVCT-L/S: isovolumetric contraction time, lateral/septal. IVRT-L/S; isovolumetric relaxation time, lateral/septal. E/A ratio; peak mitral flow velocity during early filling/peak mitral flow velocity during atrial contraction. S/D; systolic flow velocity pulmonary vein/diastolic flow velocity pulmonary vein. E/E’-L/S; peak mitral flow velocity during early filling/peak mitral annulus velocity during early filling, lateral/septal. BODY.RESULTS.IMPACT OF EXERCISE TRAINING.QUALITY OF LIFE: There was no significant change in the SF-36 total score (Table 5). There was a significant increase in the SF-36 subscale 'physical function' after training (P = 0.004, Table 5), which did not differ between groups (time*group P = 0.11). A trend for an inverse correlation was found between baseline SF-36 scores and training-induced change in SF-36 scores (r = -0.51, P = 0.052). We found no change in the MLHFQ for both groups (Table 5). No significant correlations were found between baseline MLHFQ scores and training-induced change in MLHFQ. 10.1371/journal.pone.0141256.t005 Table 5 Results of the SF-36 and Minnesota living with HF questionnaire (MLHFQ). CT (n = 9) HIT (n = 8) P-value SF-36 Pre Post Pre Post Time Group Time*Group Physical functioning 74±22 78±17 57±21 69±17 0.004 0.16 0.11 Physical health subscore † 73±20 76±16 60±22 67±19 0.15 0.26 0.52 Mental health subscore † , ‡ 81±9 83±10 83±5 82±10 0.75 0.87 0.54 Total score † , ‡ 75±16 78±13 68±14 73±14 0.18 0.42 0.76 CT (n = 9) HIT (n = 10) P-value MLHFQ Pre Post Pre Post Time Group Time*Group Total score 18±14 16±16 21±15 20±14 0.81 0.56 0.89 Data is presented as mean±SD. P-values refers to 2-way repeated measures ANOVA between the 2 training groups. Results of the SF-36 were scored on a 0–100 scale, in which a high score represents a better quality of life. Results of the MLHFQ were scored on a 0–105 scale, in which a low score indicates few HF-related complaints. † Data was available for 7 subjects in the HIT-group. ‡ Data was available for 8 subjects in the CT-group. BODY.RESULTS.IMPACT OF EXERCISE TRAINING.CONTROL GROUP: We found no changes over the 12-week period in maximal oxygen uptake (17.4±5.9 versus 17.5±5.8 ml/min/kg, P = 0.79) or in any of the other parameters of physical fitness (all P>0.05, S1 Table). Except for a decrease across time in the superficial femoral artery FMD and an increase in lateral E-E'-ratio, we found no changes in cardiac and vascular structure or function or in the SF-36 score and MLHFQ in controls (all P>0.05, S1 Table). BODY.DISCUSSION: This study comprehensively compared physical fitness, vascular function, cardiac function and quality of life between a feasible and practical HIT-protocol versus traditional CT in HF patients. We have demonstrated that CT and HIT are both feasible in HF patients and induced a significant improvement in measures of (sub)maximal exercise performance and fitness, with no significant differences between both types of exercise training. Despite these changes in fitness, we did not find improvement in measures of resting cardiac and vascular structure and function. Furthermore, except an improvement in the subscale 'physical function' that may be related to the change in fitness, no effect of training was found on quality of life. Therefore, our data suggest that both types of exercise training successfully improve measures of physical fitness within 12-weeks, but not cardiovascular function at rest or quality of life. Moreover, we found no evidence for superiority of this more feasible HIT-protocol over traditional CT on the parameters presented in our study. BODY.DISCUSSION.PHYSICAL FITNESS: Previous work found improvements in physical fitness in HF after HIT ranging from 8–46% [16,20,21], whilst studies adopting CT found changes ranging between 0–22% [17,19]. Most of these training studies have included low numbers of participants, which may explain the large variation in results. The largest HF exercise training study so far (>1,000 participants), the HF-ACTION trial, reported a median increase in fitness of 4% [39]. Although in the lower ends of the spectrum, the change in physical fitness in our study (~4%) is within the range of improvements as reported in previous work. As demonstrated in various previous studies, training characteristics (e.g. frequency, intensity and duration) are important factors determining training responses [40]. Nonetheless, our relatively low-frequency protocols were sufficient to induce significant improvements in fitness levels. Similar observations were made by Belardinelli et al [23], who adopted a long-term (10-year), low-frequency exercise training program in HF patients. They reported improvement in physical fitness levels in trained subjects after 1 year of training, whilst fitness levels remained higher than in controls across the 10-year intervention. In addition to the low frequency of training, the relatively modest changes in fitness in our study may relate to characteristics of the included participants, such as genetic factors [41] or to a priori higher levels of physical fitness as lower physical fitness levels are associated with larger training-induced improvements in HF patients [42,43]. Indeed, some previous HIT-studies demonstrated large improvement after exercise training in HF patients with low baseline levels of physical fitness [16–18]. Moreover, patients with chronotropic incompetence (i.e. 68% in our study) have attenuated exercise-induced improvement in parameters of fitness, which could have affected the effect size in our study [44]. Despite the relatively modest effect sizes, our exercise training protocols were successful in improving parameters of physical fitness. We did not demonstrate significant differences between CT and HIT on the change in physical fitness, a finding which is in agreement with some [18,19,22], but not all previous studies [16,17]. Interestingly, recent meta-analyses suggest that (high-intensity) interval training results in superior effects on physical fitness compared to CT in HF [45,46]. Despite these promising results of HIT in HF, larger trials that focus on clinical end-points are needed [47]. Also, limitations of a meta-analysis should be taken into consideration, as both HIT and CT interventions included in these meta-analyses comprise of many different protocols and exercise intensities. Especially for HIT, it is suggested that the time spent at a high percentage of peak oxygen uptake, determined by intensity and duration of the work and rest intervals, importantly contributes to the effect size [48]. Whilst the intensity of our high-intensity-bouts is high, the time spent at these high-intensity bouts was lower than in previous studies demonstrating superior effects of HIT compared to CT [16,17]. Furthermore, to validly compare exercise training regimes, it is important that total workload is not significantly different. A study that compared training-effects of HIT and CT and applied individually designed training programs with specific emphasis on comparable workloads for HIT and CT, found no differences between training modalities [19]. Therefore, more rigorous exercise programs (both in frequency, duration, and training load) rather than the type of training per se may contribute to larger improvements in physical fitness. BODY.DISCUSSION.CARDIAC AND VASCULAR ADAPTATION: Exercise in HF patients is associated with beneficial cardiac remodelling [8]. After 12-weeks of training, we found no improvements in cardiac structure and function at rest, although the increase in maximal oxygen pulse suggests an increase in stroke volume during exercise. Previous studies that reported significant changes in cardiac function or structure generally applied training periods ≥6 months [8]. Therefore, the relatively short duration of training may contribute, at least partly, to the lack of cardiac remodelling in our study. Moreover, the largest proportion of our training-participants reported ischemic HF etiology. This could be of special importance, since a recent study suggested that cardiac adaptation during outpatient rehabilitation is more prominent in HF patients with non-ischemic etiology [49]. Furthermore, our results are in line with a recent meta-analysis that could not confirm that HIT is superior to CT to induce cardiac adaptations [46]. In our study, we found no superior effect of HIT to improve vascular function, which contrasts with the findings of a recent meta-analysis [50]. However, 4 out the 7 studies included in this meta-analysis showed a superior effect of HIT. Interestingly, these studies were all from the same laboratory and did not follow contemporary guidelines to assess endothelial function. We found no overall effect of exercise training on vascular function. As previous literature has reported a wide range of exercise-induced responses (and also demonstrated non-responders) in vascular function, Green et al. investigated which factors predict this response [51]. They concluded that exercise-induced improvements in vascular function are associated with a lower pre-training vascular function. When comparing the baseline FMD values of our subjects to normal values published previously [52], the endothelial function of the subjects in our study was within the normal range for their age. This may relate to the optimal pharmacological therapy of the HF patients we included [53]. Moreover, we have previously demonstrated that the shear rate pattern during exercise (i.e. an important stimulus for exercise-induced adaptation in vascular function) is less beneficial in HF patients compared to controls [54]. Preserved FMD before training and a suboptimal shear rate stimulus during exercise training may contribute to the absence of a significant training-induced change in vascular function. BODY.DISCUSSION.QUALITY OF LIFE: Quality of life after exercise training in HF patients is previously demonstrated to improve [7,55]. In our study, we indeed found improvement in the subscale for physical functioning after exercise training, but not for total quality of life. The lack of improvement in quality of life may relate to the relatively 'good' quality of life at baseline, which was well above that of previous studies [17,56,57] and consequently, provides little space for further improvement. In support of this idea, we observed a trend for an inverse relation between baseline SF-36 and change in SF-36 in the training group. This indicates that subjects with lower quality of life prior to exercise training demonstrate a larger benefit from the intervention. Furthermore, the inclusion of more relatively old HF patients in our study may also contribute to the smaller effect size of exercise training on quality of life, as demonstrated in a previous study [58]. This latter study found that older HF patients (>60 yrs) demonstrate a smaller effect of exercise training on quality of life compared to younger HF patients (<60 yrs). BODY.DISCUSSION.CLINICAL RELEVANCE: Given the importance of (even small improvements in) fitness levels for the prognosis of HF patients [5,6,59], finding both a feasible and effective training program is clinically relevant. Another important factor is adherence to exercise training, which often is reported to be low in HF patients due to time-constraints and lack of energy [60]. A HIT training program with lower training frequency and high-intensity intervals of moderate duration might address these two major factors of non-compliance. Therefore, we have studied whether such a program is effective and whether it is superior to CT. The results of this study suggest that low frequency HIT with high-intensity intervals of moderate duration is feasible and successful in improving fitness. Such findings may be of clinical relevance and future studies should therefore focus on finding the optimal exercise protocol for HF patients to achieve long-term benefits and adherence. BODY.DISCUSSION.LIMITATIONS: Although we have included a relatively small number of patients, our sample size was in line with previous studies that demonstrated differences in effects of HIT and CT on physical fitness [16,17] and vascular function [50]. Moreover, we have used state-of-the-art techniques for measuring physical fitness and vascular function, in contrast to some previous studies that used suboptimal techniques to assess endothelial function. Therefore, we a priori expected to have sufficient power to detect significant differences between HIT and CT. Post-hoc power analysis revealed a power of 54% to detect differences in change in physical fitness between the two types of training. Finally, we did not provide a comparison between HIT with short high-intensity bouts and HIT with long high-intensity bouts. We encourage future studies to investigate whether different HIT-protocols render different results and to elucidate the optimal HIT-protocol. In conclusion, we have demonstrated that 12-weeks of exercise training in HF patients is associated with improvements in parameters of physical fitness, whilst no improvements in cardiovascular function at rest or (total) quality of life are observed. Moreover, our data does not provide strong evidence for a potentially superior improvement in physical fitness, cardiovascular function or quality of life after 12-weeks of HIT compared to CT in HF patients NYHA class II-III. BODY.SUPPORTING INFORMATION: S1 TREND ChecklistTREND statement checklist.(PDF)Click here for additional data file. S1 ProtocolResearch protocol.(PDF)Click here for additional data file. S1 TableResults of the control group.Data is presented as mean ±SD. †Data missing for 1 control subject. VO2peak; peak oxygen uptake. AT; anaerobic threshold. BA; brachial artery. SFA; superficial femoral artery. GTN; glyceryl trinitrate. CADC; conduit artery dilating capacity. IMT; intima-media thickness. SRAUC; shear rate area-under-the-curve. CADC; conduit artery dilating capacity. LVEDV; left ventricular end-diastolic volume. LVESV; left-ventricular end-systolic volume. IVCT-L/S: isovolumetric contraction time, lateral/septal. IVRT-L/S; isovolumetric relaxation time, lateral/septal. E/A ratio; peak mitral flow velocity during early filling/peak mitral flow velocity during atrial contraction. S/D; systolic flow velocity pulmonary vein/diastolic flow velocity pulmonary vein. E/E'-L/S; peak mitral flow velocity during early filling/peak mitral annulus velocity during early filling, lateral/septal. MLHFQ; Minnesota living with heart failure questionnaire.(DOCX)Click here for additional data file.

TITLE: Comparing the Hemodynamic Effects of Spinal Anesthesia in Preeclamptic and Healthy Parturients During Cesarean Section ABSTRACT.BACKGROUND: Despite controversies about the safest anesthetic technique for cesarean delivery in severely preeclamptic women, there is evidence that supports the use of spinal anesthesia in this group of patients. ABSTRACT.OBJECTIVES: This prospective randomized clinical trial was designed to determine the hemodynamic effects of low-dose spinal bupivacaine and the incidence of spinal anesthesia-associated hypotension in severely preeclamptic and healthy parturients undergoing cesarean sections. ABSTRACT.PATIENTS AND METHODS: Spinal anesthesia was performed with 10 mg (= 2 mL) hyperbaric 0.5% bupivacaine plus 2.5 μg sufentanil in two groups of patients after they received 500 mL of IV lactated Ringer's solution. Heart rate and blood pressure were recorded before spinal anesthesia and at two minutes intervals for 15 minutes after the block, and then every five minutes until the end of the surgery. Hypotension was defined as more than 25% of decline in the mean arterial blood pressure compared to the baseline in both groups (or systolic blood pressure < 100 mmHg in healthy parturients) and was treated with 5 mg IV ephedrine. The total amounts of intravenous administered fluid and the total doses of ephedrine were recorded for each patient as well. ABSTRACT.RESULTS: The incidence rate of hypotension among the preeclamptic patients was lower than that of the healthy parturients, despite the former group receiving smaller volumes of intravenous fluids (P < 0.05). The total doses of IV ephedrine for treating hypotension were significantly lower among the preeclamptic patients (3.2 mg in preeclamptic patients versus 7.6 mg in normotensive patients) (P = 0.02). The one-minute Apgar score was significantly lower for the preeclamptic parturients (8.4 ± 0.7 versus 7.2 ± 1.5) (P = 0.001), but there was no significant difference in the five-minute Apgar scores between the two groups. ABSTRACT.CONCLUSIONS: Our results confirm that low-dose bupivacaine spinal anesthesia is associated with a lower risk of hypotension than previously believed, and it can therefore be safely used in severe preeclamptic women undergoing cesarean delivery. BODY.1. BACKGROUND: Pregnancy-induced hypertension is a major cause of morbidity and mortality in obstetrics, complicating 3% - 8% of pregnancies. Severe preeclampsia poses a dilemma for anesthesiologists, and there is some controversy about the best anesthetic technique for cesarean delivery in such cases (1, 2). Because of the risks related to airway edema, difficulty with the airway or failed intubation, hypertensive response to direct laryngoscopy, and aspiration pneumonitis, general anesthesia is associated with more untoward outcomes in this particular group of patients (3, 4). When there is no contraindication for performing regional anesthesia, risk-benefit considerations strongly favor neuraxial techniques over general anesthesia for cesarean delivery in cases of severe preeclampsia. Regional anesthesia techniques have been widely used recently, however, spinal anesthesia, once considered contraindicated due to the common belief that the sudden and extensive sympathetic blockade following the subarachnoid block would result in severe hypotension and compromise uteroplacental blood flow in this group of patients (5-8). Although controversial, some studies have shown the effectiveness of colloid loading on reducing the incidence of hypotension in spinal anesthesia (9, 10), but vasopressor agents and volume loading, which are commonly used to manage spinal anesthesia-induced hypotension, could put the preeclamptic patients at increased risk of hypertension and pulmonary edema (6). Recent evidence has challenged this view, suggesting that spinal anesthesia may in fact be an appropriate choice for preeclamptic women when cesarean delivery is planned, as long as neuraxial anesthesia is not contraindicated (e.g., coagulopathy, eclampsia with persistent neurologic deficits) (2, 5, 8). Although the relative safety of the subarachnoid block in these patients has been demonstrated, there are few studies that compare the differences in the hemodynamic changes and newborn well-being after single-shot spinal anesthesia between preeclamptic and healthy parturients BODY.2. OBJECTIVES: This prospective randomized clinical trial was designed to compare the hemodynamic effects and the incidence of spinal anesthesia-associated hypotension after spinal anesthesia with bupivacaine plus sufentanil in severely preeclamptic versus healthy parturients undergoing cesarean sections. BODY.3. PATIENTS AND METHODS: After obtaining institutional ethics committee approval, 80 parturients (37 healthy and 43 severely preeclamptic parturients) that were being cared for in our unit from April 2011 to July 2012 were enrolled in the study after providing informed consent. Severe preeclampsia was defined as a systolic arterial blood pressure of 160 mmHg or higher, or a diastolic blood pressure of 110 mmHg or higher, associated with proteinuria > 5 g in 24 hours. Patients who were excluded were those with coagulopathy (including those with platelet counts < 50,000), placental abruption, severe fetal distress, a history of allergy to local anesthetics, oliguria of less than 500 mL in 24 hours or persistently < 30 mL/hour, cerebral or visual disturbances, pulmonary edema, hemodynamic instability, local infection of the spinal injection site, or refusal of a spinal block. All patients in the preeclampsia group received a 4 g loading dose of intravenous magnesium sulfate (Mg SO4) followed by a 1 g/hour infusion for 24 hours for seizure prophylaxis. Intravenous hydralazine of 5 mg was given at 20-minute intervals to decrease diastolic blood pressure to approximately 90 mmHg. Before performing spinal anesthesia on each patient, preoperative fluid administration equal to 10 mL/kg of Ringer's lactate solution was administered over the course of 15 - 20 minutes. All patients received 1500 - 2000 mL lactated Ringer's solution after spinal anesthesia and during the operation. The volume of administered fluid was not restricted in the preeclamptic patients because of the contracted intravascular volume in this group of patients and the high incidence of hypotension caused by spinal anesthesia-induced sympathetic blockade. Patients were monitored with standard monitoring devices including automated blood pressure cuffs, electrocardiogram, and pulse oximetry. Spinal anesthesia was performed with 10 mg hyperbaric 0.5% bupivacaine plus 2.5 - 3 μg sufentanil (2.5 mL volumes) in two groups in the sitting position in the L3-L4 or L4-L5 vertebral interspaces. Each patient was then placed in the supine position with a left lateral tilt of 15-20 degrees. The height of the sensory block was assessed using a pinprick test, and a 10°-15° head down-tilt (Trendelenburg position) was initiated if a T4 sensory level was not achieved at 10 minutes after the spinal injection. After achieving an adequate sensory block (T4 level), the patient was prepared for surgery. Heart rate and blood pressure were recorded before performing spinal anesthesia at two-minute intervals for 15 minutes after the block, and then every five minutes until the end of the surgery. Hypotension was defined as more than 25% decline in mean arterial blood pressure (MAP) compared to the baseline in both groups (or systolic blood pressure (SBP) < 100 mmHg in healthy parturients) and was treated with 5 mg IV ephedrine. The total amounts of intravenous administered fluid and the total doses of ephedrine were recorded as well. Based on the findings of previous studies, we calculated that at least 38 patients per group were required to show a 25% difference in the incidence of hypotension, with 80% power at the 5% level. Data are presented as number, median and range, mean ± SD, or percentage as appropriate. Fisher's exact test was used for intergroup comparisons of the incidence of hypotension. Mean values of most of the quantitative study variables were compared by using the unpaired Student's t-test. A P value of less than 0.05 was considered to indicate statistical significance. Data entry and analysis were performed using SPSS software version 16. BODY.4. RESULTS: Eighty patients (severe preeclampsia = 43 and healthy = 37) were studied. Demographic data, the times from spinal injection to delivery, the median sensory blocked levels at the time of incision, the volumes of estimated blood loss, and the surgical durations were similar between the two groups (Table 1). Table 1. Maternal, Neonatal, and Anesthetic Considerations a Variable Healthy Preeclampsia P Value n 37 43 Age, y 28.1 ± 5 29.3 ± 6.6 0.12 Gestational age, week 38.8 ± 1.2 34.3 ± 3.8 0.00 Base line MAP 101.1±10.1 119.5±15.9 0.02 Upper Sensory Level (Median, Range) T4 (T2-T5) T4 (T2-T5) 0.32 IV fluid, mL 2500 ± 0.1 2400 ± 0.2 0.000 Spinal puncture to delivery interval, minute 17.5 ± 2.5 17.1 ± 2.5 0.83 Ephedrine dose, mg 7.5 ± 9.2 3.2 ± 7.8 0.04 APGAR score 1-min (Range) 8.4 ± 0.7, (5 - 10) 7.2 ± 1.5, (2 - 10) 0.001 APGAR score 5-min (Range) 9.4 ± 0.7, (7 - 10) 8.6 ± 1.1, (5 - 10) 0.35 a Values are expressed as mean ± SD unless otherwise indicated. The mean gestational age and mean one-minute Apgar scores in the patients with severe preeclampsia were significantly lower than those of the healthy parturients (Table 1). However, there was no significant difference in the five-minute APGAR score between the two groups. The incidence rate of hypotension in the preeclamptic patients (55.8%) was less than that of the healthy parturients (89.2%) (Table 2), despite the former receiving smaller volumes of intravenous fluids (2.4 versus 2.5 lit) (Table 1) (P = 0.001). Table 2. Incidence of Hypotension and Changes in Blood Pressure After Spinal Anesthesia in the Two Groups Variable Healthy Preeclampsia P Value Incidence of MAP hypotension (%) 33 (89.2%) 24 (55.8%) 0.001 Lowest SBP mmHg 98 ± 12.9 116 ± 18.6 0.02 Lowest DBP mmHg 47.5 ± 9.5 66.3 ± 14.1 0.03 Lowest MAP mmHg 64.9 ± 10.1 83.7 ± 14.9 0.008 The SBP, DBP, and MAP measured at the baseline were higher for the patients with preeclampsia, and the mean lowest SBP and MAP measured among the preeclamptic patients were consistently higher than the corresponding values among the healthy parturients (Table 2). Furthermore, the total doses of IV ephedrine for treating hypotension were significantly lower for the preeclamptic patients than for the normotensive patients (3.2 ± 7.8 mg versus 7.5 ± 9.2 mg) (P = 0.04). BODY.5. DISCUSSION: Spinal anesthesia-associated hypotension may occur in up to 64% - 100% of pregnant women undergoing cesarean delivery (2). Severely preeclamptic patients have been considered to be at higher risk of severe hypotension (1, 2, 5-8), and the concern of severe hypotension caused by subarachnoid block may often deter the anesthesiologist from choosing this technique for this group of patients. Epidural anesthesia has traditionally been regarded to be safer for preeclamptic parturients as it does not produce sudden hypotension. However, some studies have shown that the two techniques produce a similar incidence and severity of hypotension in preeclamptic parturients (6, 7, 11). There is growing interest in using spinal anesthesia on preeclamptic patients because of its simplicity, faster onset, lower dose of injected local anesthetic (which decreases the probability of systemic toxicity), and less tissue trauma caused by the use of a smaller gauge spinal needle (12-14). As a result of this interest, a number of studies have been conducted to show the hemodynamic consequences of spinal anesthesia in patients with preeclampsia. A prospective study by Aya et al. found that the risk of hypotension following spinal anesthesia in preeclamptic patients was significantly lower than the risk among healthy term parturients (17% in preeclamptic parturients and 53% in healthy parturients) (2). In another study, the same author suggested that the risk of hypotension following a subarachnoid block in preeclampsia was related to preeclampsia-associated factors rather than a small uterine size (15). Similar to the studies by Aya et al., the incidence of hypotension in severely preeclamptic patients undergoing spinal anesthesia for cesarean delivery was found to be significantly lower in comparison to the rate among healthy parturients (55% versus 89%) in our study. Factors such as difference in gestational age, the carrying of a smaller fetus, less aortocaval compression, sympathetic hyperactivity, and high vascular tone might have led to this finding (1, 2, 8, 16). The injection of different doses of bupivacaine (10 mg versus 8 - 12 mg) for the induction of the subarachnoid block and the different criteria for defining hypotension (a 25% versus 30% decline to baseline MAP) might explain why the incidence of hypotension was higher in both groups in our study compared to the corresponding rates in Aya et al.'s study. According to two other studies conducted by Mendes et al. (17) and Saha et al. (8), the hemodynamic changes and newborn well-being appeared to be comparable in severely preeclamptic and healthy parturients submitted to spinal anesthesia for cesarean section, and spinal anesthesia seemed to be a safe option for patients with severe preeclampsia. The ephedrine requirement for treatment of spinal anesthesia-induced hypotension in preeclampsia has been reported to be lower than that required by healthy parturients (2, 18). Preeclamptics have been reported as requiring significantly less phenylephrine to treat hypotension as well (8). These results were comparable to our findings, in that the total doses of IV ephedrine for treating hypotension were significantly lower for the preeclamptic patients (3.2 ± 7.8 mg) than for the normotensive patients (7.5 ± 9.2 mg) (P = 0.04). There is little evidence in the current literature supporting the use of phenylephrine as the vasopressor of choice in high-risk pregnancies such as those involving preeclampsia (19), so we chose to use ephedrine for treating hypotension in our patients. More studies are needed to investigate the effects of vasopressors while considering the influence on feto-maternal physiology in patients with preeclampsia. The results of a review by Dyer et al. showed that after spinal anesthesia for cesarean section, patients with preeclampsia had a lower susceptibility to hypotension and less impairment of cardiac output than healthy parturients (20). In a prospective observational study on 15 parturients with severe preeclampsia, no clinically significant change in cardiac output was shown after the subarachnoid block (21). The focus of our study was on the blood pressure changes during spinal anesthesia in the preeclamptic patients, and therefore we did not measure the cardiac output fluctuations in our patients. Further studies with larger sample sizes evaluating cardiac output are needed for better understanding of hemodynamic changes during spinal anesthesia in this group of patients. It is believed that the incidence of spinal anesthesia-induced hypotension is related to the local anesthetic dose, so one particular strategy to minimize the hemodynamic disruption after spinal anesthesia involves using small intrathecal local anesthetic doses. In a pilot study which compared the hemodynamic consequences of two doses of spinal bupivacaine (7.5 mg versus 10 mg) for cesarean delivery in those with severe preeclampsia, pre-delivery MAP was lower and the ephedrine requirements were greater in the 10 mg group (22). In another study, Roofthoof and Van de Velde have shown that when low-dose spinal anesthesia (6.5 mg bupivacaine) was administered with sufentanil as part of a combined spinal-epidural (CSE) technique in shorter surgeries (less than 60 minutes), the need for epidural supplementation was rare (23). The ED95 of intrathecal bupivacaine coadministered with intrathecal 2.5 μg sufentanil using CSE anesthesia for cesarean section in severely preeclamptic patients was reported to be 8.82 mg in another study, and using smaller doses of intrathecal bupivacaine in the patients resulted in a decrease of incidences of maternal hypotension and vasopressor requirements (24). However, no studies have compared CSE with single-shot spinal anesthesia in severe preeclampsia, and further research is needed to elucidate the best strategy to optimize the hemodynamics and uteroplacental perfusion in this particular group of patients. Considering the neonatal outcomes after various anesthesia techniques in cesarean delivery among preeclamptic patients, no statistically significant difference was found in the one- and five-minute Apgar scores and the umbilical artery blood gas markers between the two groups of patients receiving spinal or general anesthesia (25). Other studies in support of subarachnoid block have also shown that transient neonatal depression and birth asphyxia are more common among preeclamptic women who have received general anesthesia (26). Comparing umbilical arterial fetal base deficit and other markers of maternal and neonatal well-being in 70 preeclamptic patients undergoing cesarean delivery who were randomized into groups receiving either spinal or general anesthesia, the spinal group had a higher mean umbilical arterial base deficit and a lower median umbilical arterial pH, but other markers of a compromised neonatal condition, including the requirement for neonatal resuscitation, an Apgar score < 7, an umbilical arterial pH < 7.2, and the need for neonatal intermittent positive pressure ventilation were the same among the two groups (27). In comparison with healthy subjects, patients with severe preeclampsia had a younger gestational age (34 weeks versus 39 weeks) in our study, which is one of the likely causes of the lower one-minute Apgar scores of the neonates among the first group. Although there was evidence as early as 1950 that preeclampsia attenuates spinal anesthesia-induced hypotension, it has taken a long time for clinical trials to demonstrate the safety of spinal and CSE anesthesia in preeclamptic parturients. Recently, after five decades of research, the relationship between spinal anesthesia, pre-eclampsia, and hypotension can be properly acknowledged and put into clinical practice (28). Because of an altered balance of vascular tone, reduced responses to endogenous pressors, and increased synthesis of vasodilator prostaglandins and nitric oxide, the normal pregnant patient is very sensitive to spinal anesthesia. These effects increase dependence on sympathetic vascular tone in normal pregnancy, and this can be the main cause of spinal anesthesia-induced hypotension in healthy parturients, while damaged vascular epithelium results in persistent vasoconstriction in preeclampsia (8, 16). There is a dramatic increase in the use of spinal anesthesia for cesarean delivery in severe preeclampsia that could be related to the documented safety of subarachnoid block in this group of patients. Therefore, single-shot subarachnoid block may be a good choice for cesarean delivery in patients with severe preeclampsia, since it has been shown to be safe for both the mother and the neonate (28). BODY.5. DISCUSSION.5.1. CONCLUSION: Our results have also confirmed that single-shot low-dose bupivacaine spinal anesthesia is associated with a lower risk of hypotension and vasopressor requirements in comparison to the rates of healthy subjects, and could be safely used in patients with severe preeclampsia undergoing cesarean delivery. However, more studies with the CSE technique using smaller doses of local anesthetics and larger sample sizes are suggested. Further research is needed to find the best strategies to optimize hemodynamics and uteroplacental perfusion in severely preeclamptic parturients during spinal anesthesia for cesarean delivery.

TITLE: Efficacy and Safety of Tiropramide in the Treatment of Patients With Irritable Bowel Syndrome: A Multicenter, Randomized, Double-blind, Non-inferiority Trial, Compared With Octylonium ABSTRACT.BACKGROUND/AIMS: Antispasmodics such as octylonium are widely used to manage irritable bowel syndrome (IBS) symptoms. However, the efficacy and safety of another antispasmodic, tiropramide, remain uncertain. We aimed to evaluate the efficacy and safety of tiropramide compared with octylonium in patients with IBS. ABSTRACT.METHODS: In this multicenter, randomized, non-inferiority trial, 287 patients with IBS (143 receiving tiropramide and 144 octylonium) were randomly allocated to either tiropramide 100 mg or octylonium 20 mg t.i.d (means 3 times a day) for 4 weeks. Primary endpoint was the mean change of abdominal pain from baseline assessed by visual analogue scales (VAS) score after 4 weeks of treatment. Secondary endpoints were the changes in abdominal pain from baseline at week 2 and in abdominal discomfort at weeks 2 and 4, using VAS scores, patient-reported symptom improvement including stool frequency and consistency, using symptom diaries, IBS-quality of life (IBS-QoL), and depression and anxiety, at week 4. ABSTRACT.RESULTS: The VAS scores of abdominal pain at week 4, were significantly decreased in both tiropramide and octylonium groups, but the change from baseline did not differ between the 2 groups (difference,-0.26 mm; 95% CI,-4.33-3.82; P = 0.901). Abdominal pain and discomfort assessed using VAS scores, diaries, and IBS-QoL were also improved by both treatments, and the changes from baseline did not differ. The incidence of adverse events was similar in the 2 groups, and no severe adverse events involving either drug were observed. ABSTRACT.CONCLUSIONS: Tiropramide is as effective as octylonium in managing abdominal pain in IBS, with a similar safety profile. BODY.INTRODUCTION: Irritable bowel syndrome (IBS) is a functional bowel disorder characterized by abdominal pain or discomfort relieved by defecation and accompanied by a change in bowel habit.1 According to epidemiologic studies in Korea, the prevalence of IBS was reported to be 6.6% overall, 7.1% in men and 6.0% in women.2 Patients with IBS are reported to seek healthcare frequently, have poor health-related quality of life (QoL) and be responsible for high societal costs.3-5 Like other functional gastrointestinal (GI) disorders, the development of IBS and its symptoms are associated with multifactorial pathophysiology.6 Moreover, symptoms even in an individual patient with IBS may have a different pathophysiology depending on various factors such as diet or emotional status. Therefore, therapy targeting a specific pathophysiology in IBS has been difficult, and symptom treatment focusing on major complaints of IBS or drug combination covering multiple pathophysiology has usually been applied to the management of IBS. Of multifactorial pathophysiology in IBS, colonic smooth muscle spasm has long been postulated to be responsible for abdominal pain in IBS from clinical observations as well as some experimental evidences.7 Therefore, pharmacologic agents directly acting on smooth muscle or on cholinergic receptors, called as antispasmodics, have been developed and used to control abdominal pain and discomfort in IBS. They may be classified as smooth muscle relaxants or agents with anti-cholinergic or anti-muscarinic property. One of the antispasmodics, tiropramide, is a derivative of tyrosine, and exerts a spasmolytic effect on the intestine through reducing Ca2+ release into intestinal smooth muscle, rather than acting on the enteric nervous system.8,9 In a double-blind, placebo-controlled, randomized trial, tiropamide led to symptom improvement in terms of total symptom scores for 4 weeks, compared with 3 weeks in the placebo group; in addition, at week 4 abdominal pain was only improved in the tiropramide group.10 Also in a Korean study of similar design involving 63 patients with IBS, tiropramide improved overall symptom scores and decreased abdominal pain at weeks 2 and 4 (78.2% of the treatment group with tiropramide and 40.5% of placebo group).10 Another antispasmodic, octylonium, also modifies Ca2+ fluxes in the intestinal smooth muscle, with direct myolytic properties similar to tiropramide.11,12 Previously, octylonium has already been proven to be more effective in reducing IBS symptoms than placebo.13-15 However, there have been few large-scale clinical trials comparing tiropamide with antispasmodics of proven efficacy in IBS patients. Therefore, this study aimed at evaluating the efficacy and safety profile of tiropamide in IBS patients compared with octylonium. BODY.MATERIALS AND METHODS.STUDY PATIENTS: Eligible patients were 20 to 75 years of age; had been diagnosed with IBS (as assessed according to the Rome III diagnostic criteria for IBS); complained of abdominal pain or discomfort at least 2 times per week for the 2-week run-in period; had visual analogue scale (VAS) scores over 30 mm for abdominal pain during the randomization period. Patients who fit the study criteria had a run-in period of at least 2 weeks during which all IBS-related medications, and concomitant medications were stopped. Exclusion criteria included a history of inflammatory bowel disease, lactose intolerance, uncontrolled diabetes (haemoglobin A1C > 8%) or hypertension (systolic or diastolic blood pressure of ≥ 140/90 mmHg), psychiatric disorder requiring medication, malignancy of organs other than digestive (except for cases with no recurrence for over 5 years since treatment), laparoscopic or open abdominal surgery (except for appendectomy), renal or hepatic disease, glaucoma, and prostate hyperplasia. Those with a history of the following diseases within the previous 6 months were also excluded; peptic ulcer disease, GI bleeding, gastroesophageal reflux disease, intestinal stenosis or obstruction, infectious diarrhea, and pancreatic insufficiency. Patients were excluded if they were intolerable of or sensitive to, study drug, had a history of alcohol or drug addiction, or were expected to take any contraindicated medication during the study period. Pregnant or lactating women and women of childbearing age not using contraception, were also excluded. BODY.MATERIALS AND METHODS.STUDY DESIGN: This was a multicenter (20 centers), randomized, double-blind, parallel-group, non-inferiority comparative study conducted in South Korea from May 2012 to April 2013. The study protocol was approved by the Institutional Review Board of each study center and conducted in accordance with the ethical principles based on the Declaration of Helsinki and Good Clinical Practice guidelines. For a run-in period of about 2 weeks, eligible subjects for screening were instructed to stop IBS-related medications and prohibited from other drugs. Following the screening phase of 2 to 4 weeks, eligible patients were randomly assigned to either tiropramide (Tiropa; Dae Woong Pharma Korea Inc., Seoul, South Korea) 100 mg or octylonium (Menoctyl; DONG HWA PHARM, Seoul, South Korea) 20 mg, taken 3 times daily before meals for 4 weeks. Treatment assignments were carried out by a computer-generated randomization schedule that was designed to allocate patients to the 2 treatment arms in a 1:1 ratio. The subjects were assigned to sequential allocation numbers at each site, and the medications were presented as 2 identically-appearing tablets containing 1 active drug and 1 placebo of each comparative drug to maintain the double blind condition. Follow-up visits during the study period were scheduled at weeks 2 and 4 to assess abdominal pain/discomfort using VAS, IBS quality of life, IBS symptoms including bowel habits using diaries, the Beck depression inventory (BDI) and the Beck anxiety inventory (BAI), drug compliance and adverse events (Fig. 1). Written, informed consent was obtained from all subjects before enrollment. BODY.MATERIALS AND METHODS.STUDY ASSESSMENTS.EFFICACY: The primary endpoint was the change in abdominal pain from baseline assessed by VAS scores after 4 weeks of treatment. The secondary endpoints included the change in abdominal pain from baseline by VAS scores at week 2, the change in abdominal discomfort from baseline by VAS scores at weeks 2 and 4, improvement of patients' overall assessment at weeks 2 and 4, the change in IBS QoL from baseline at 4 weeks, the change in symptom improvement in abdominal pain and discomfort from baseline using symptom diaries (severity of symptoms and days of no symptoms), and stool frequency and form assessed by Bristol stool form scale and according to IBS subtypes. Symptom improvement was defined as "completely improved," "considerably improved" or "slightly improved." Percentages of symptom improvement include "completely, considerably and slightly improved." Symptom diaries assessed daily symptoms of abdominal pain and discomfort using VAS of 0 to 100 with 100 representing extreme symptom. The number of defecation per day and the most common stool consistency during the day were recorded using the Bristol stool form scale. IBS-QoL was assessed by asking patients to answer each question using a 5-point Likert scale (0 = none, 1 = mild, 2 = moderate, 3 = severe, 4 = extremely severe). Exploratory end points were the changes in BDI and BAI from baseline. Each BDI or BAI was composed of 21 questions and assessed as "0 = do not feel at all," "1 = feel a bit," "2 = feel significantly," and "3 = feel badly." Confounding factors were baseline factors of efficacy including BDI and BAI. BODY.MATERIALS AND METHODS.STUDY ASSESSMENTS.SAFETY: Safety assessments included adverse events, and abnormalities in laboratory findings and vital signs. BODY.MATERIALS AND METHODS.STATISTICAL METHODS: We estimated the required sample size for this study from the data of a previous study which found a change in VAS for abdominal pain from baseline as 23.6 mm with standard deviation (SD) of 25.0 mm.14 The margin of non-inferiority was assumed to be 11 mm, approximately half of 22.7 mm, which was the difference of the changes observed between the octylonium and the placebo groups. Based on these assumptions, a sample of 129 patients was needed for each study arm in order to have a power of 90%, and α of 0.025 (one-sided), while allowing for a 15% dropout rate. The analyses were performed using full analysis set (FAS) and per protocol set (PPS). All efficacy analyses were conducted in the FAS population, which consisted of all randomized patients satisfying the inclusion and exclusion criteria who received at least one dose of study medication and had at least one post-baseline efficacy measurement. The primary efficacy analysis was based on the PPS, which included all patients within the FAS population who took more than 80% of their assigned drugs and had no major protocol violations such as violations of scheduled visits or administration of contraindicated medication. The change in VAS for abdominal pain from baseline was evaluated by 2 sample t test. Seconday outcomes were compared by 2 sample t test or Wilcoxon's rank sum test. Rates of symptom improvement were described as frequencies and percentages, and compared between groups by Pearson's chi-square test or Fisher's exact test. For safety variables including treatment emrgent adverse event (TEAE), adverse drug reaction, and serious adverse event, Pearson's chi-square test or Fisher's exact test was used to evaluate differences in incidence between groups. Changes in continuous variables from baseline after treatment were analyzed by 2 sample t test or Wilcoxon's rank sum test, while categorical variables, by Pearson's chi-square test or Fisher's exact test. Confounding factors such as BDI, BAI and baseline values of the efficacy variables were further analysed by ANCOVA or multiple logistic regression. A P-value of 0.05 was considered statistically significant. Statistical analyses were performed using SAS (Enterprise Guide 4.3). BODY.RESULTS.BASELINE CHARACTERISTICS: Total of 356 patients were evaluated for study inclusion. After exclusion of 61 patients during the screening period, 295 patients were randomized to either tiropramide or octylonium. Three percent of patients in the tiropramide group (4/143) and octylonium group (4/144) did not complete the study. Data on the remaining 287 patients were available for the FAS analysis: 143 for tiropramide vs. 144 for octylonium. Data on 245 patients were available for the PPS analysis: 117 for tiropramide vs. 128 for octylonium. A flowchart of patient progression through the study with reasons for premature discontinuation is presented in Fig. 2. Demographic and baseline characteristics of patients are presented in Table 1. Both treatment arms were well balanced for gender, age and severity of symptoms at baseline. BODY.RESULTS.EFFICACY: The PPS population comprised of 245 patients (117 in the test group and 128 in the control group). The mean changes of VAS scores for abdominal pain from baseline were-23.49 ± 17.51 mm in the tiropramide group and-23.13 ± 17.61 in the octylonium group. The mean difference between the 2 groups was-0.26 mm in the FAS and-0.35 mm in the PPS, and the upper limit of the 97.5% CI at one-sided was 3.82 mm in the FAS and 4.07 mm in the PPS; these are below the margin of inferiority of 11 mm, indicating that tiropramide is not inferior to octylonium (Table 2). In secondary outcome analyses, VAS scores for abdominal pain at week 2 were significantly decreased in both groups (Table 3). Significant improvement in abdominal pain and discomfort at weeks 2 and 4 using symptom diaries, and in IBS-QoL, BDI and BAI at week 4 were observed in both groups, however the differences between the groups were not significant. In the subgroup of IBS-D, stool frequency was decreased and stool consistency was improved by treatment. Furthermore, the degree of symptom reduction in abdominal pain using symptom diaries was higher in the tiropramide group than in the octylonium group from week 1 (P = 0.033). After adjusting the baseline values of related factors, our analysis showed that all the factors based on symptom diaries, except the change of IBS symptoms, did not differ in the 2 groups. IBS symptoms at week 4 using symptom diaries were reduced further compared to baseline in the tiropramide group than the octylonium group. BODY.RESULTS.SAFETY: Adverse events reported during the study are shown in Table 4. TEAE was 10.88% (16/147) in the tiropramide group and 12.24% (18/147) in the octylonium group, which included 28 each. The safety profile of tiropramide was similar to that of octylonium. Adverse drug reactions were reported in 7 patients in the tiropramide group and 6 patients in the octylonium group, which included 17 and 9, respectively. Serious adverse events developed in one patient in each group, idiopathic thrombocytopenic purpura in the tiropramide group and intervertebral disc protrusion in the octilonium group, but were not related to the study drugs, and were improved without any sequelae. Laboratory test results, when comparing those at screening period with those at last visit, showed no violation of safety regarding normal values, or the changes of direction and quantity. Vital signs did not change significantly from baseline in either group at weeks 2 and 4. BODY.DISCUSSION: The present study was designed to assess the non-inferiority of tiropramide compared to the proven antispasmodic, octylonium, for managing IBS symptoms. This multicenter, randomized, double-blind, non-inferiority trial showed that 4-week treatment with tiropramide improved abdominal pain and discomfort, patient-reported overall assessments of IBS symptoms, IBS-QoL, depression and anxiety, with no significant differences from octylonium. These results demonstrate non-inferiority of tiropramide in efficacy and safety, compared with otiolonium. Tiropramide effectively and safely managed abdominal pain in the IBS patients. Our results showed that the change in VAS scores for abdominal pain from baseline was about 23.5 mm after 4-week treatment with tiropramide, similar to 23.6 mm observed after 4-week treatment with octylonium. The changes from baseline between the 2 groups were not significantly different, and the upper limit of 95% CI was lower than the margin of inferiority in both the FAS and PPS populations, indicating non-inferiority of tiropramide compared to octylonium. In a previous study comparing tiroparmide with octylonium in IBS patients, the proportion of patients whose abdominal pain was relieved by treatment was higher in tiropramide group than in octylonium group at days 3 and 5 of medication, suggesting rapid drug action of tiropramide.16 Also, at the end of 30-day treatment, normal gut motility was restored in 88% of the tiropramide group compared with 47% of octylonium group.16 The presumed mechanism of tiropramide in improving abdominal pain is its antispasmodic effect on the intestine. In an animal study using the isolated smooth muscle, tiropramide inhibited Ca2+ influx for the most part and, to a lesser extent, increased the intracellular cyclic adenosine monophosphate (cAMP) levels.9 This study using rabbit also found that tiropamide increased Ca2+ absorption into microvesicles and decreased the amount of Ca2+ released to stimulate the contraction of the smooth muscle by increasing cAMP; as a consequence the smooth muscle relaxed.9 In terms of the distribution of tiropramide, a study showed that the radioactivity of tiropramide lasted longer in the colon than in other organs after intravenous or oral administration,17 indicating relative colonic selectivity. Furthermore, the effect of tiropramide were more evident in the smooth muscle of the digestive, urinary and female genital tract than in those of the vessel, and in stimulated contractions rather than in spontaneous ones.17 In the present study, other advantages of tiropramide were observed compared with octylonium: tiropramide was effective earlier than octylonium; in the assessment using symptom diaries, the severity and degree of abdominal discomfort tended to be reduced further in the tiropramide group than in the octylonium group. Since this study was designed to evaluate the non-inferiority of tiropramide over octylonium, we could not conclude that tiropramide is more effective than octylonium, but we may at least speculate about this possibility, considering that assessment using symptom diaries is less biased than that using VAS, which is based on patients' rememberance of symptoms from the previous week. Taken together, there may be some advantage of tiroparmide over octylonium for managing IBS symptoms; this could be due to its colonic selectivity. Regarding the safety of tiropramide, percentages of patients reporting adverse events were similar in both groups. Vital signs and labortory test results did not significantly change after a 4-week treatment with either drug. It has been known that it does not affect neurotransmitters such as histamine, acetylcholine, and adrenaline, thus leading to a lower incidence of side effects than other antispasmodic agents. It was also found that tiropramide did not affect esophageal motility, including the pressure of the low esophageal sphincter, and the coordinated movements of the esophagus.18 Actually, both tiropramide and octylonium have been safely prescribed over the past ten years. Similarly, the present study raised no safety concern. In the subgroup of patients with diarrhea predominant IBS (IBS-D), tiropramide improved bowel habits. Its spasmolytic effect of the drug may be related to the improvement of bowel habits in terms of stool frequency and consistency. These improvements may also be explained by the fact that tiropramide can restore normal intestinal transit in patients with IBS-D having rapid intestinal transit, and are consistent with observations in a study on octylonium demonstrating improvements of bowel habit and abdominal pain and discomfort.14 Moreover, as previously mentioned, gut motility was normalized in 88% of the tiropramide group after 30-day treatment with tiropramide, against 47% of the octylonium group.16 Considering the effect on bowel habit improvement in the subgroup of patients with IBS-D, there may be an additional benefit of tiropramide in patients with IBS-D, although the small number of patients with constipation predominant IBS (IBS-C) in our study might preclude us from concluding that the study drug is effective in patients with IBS-C. In this study, levels of depression and anxiety in the IBS patients were also improved by tiropramide. This may be interpreted in terms of the brain-gut axis.19 Relief of abdominal pain may affect positively the emotional status of study patients. IBS-QoL was also improved. However, theses secondary outcomes should be cautiously interpreted because there may be a variety of confounding factors. Placebo effects are known to be relatively high in IBS, ranging from 20% to 70%. This is believed to be partly due to a variety of physical and psychological factors affecting the brain-gut axis, which is thought to be involved in IBS pathophysiology. Therefore, studies of the treatment of IBS should be designed as randomized, double-blind, and placebo-controlled trials.20 The present study lacks a placebo control therefore could be limited in directly showing the superiority of tiropramide to placebo. However, this limitation in our study may be sufficiently compensated by previous evidence to demonstrate the superiority of octylonium to placebo.13,14,21 Other limitations of our study include the relatively short period of treatment (only 4 weeks) and the lower dose of octylonium than that used in the reference study (20 mg vs. 40 mg, t.i.d). However, the efficacy of both tiropramide and octylonium in improving symptoms of IBS was clearly demonstrated by our study results with statistical differences between pre-and post-treatments. In conclusion, our study demonstrates that treatment with tiropramide is effective and safe for managing IBS symptoms such as abdominal pain and discomfort, stool frequency and consistency, IBS-QoL, depression and anxiety. Considering not enough data fully show the efficacy and safety of tiropramide in IBS patients, the present study should be a useful reference in IBS research and clinical practice for managing IBS patients. Further studies are required to elucidate the mechanism of action of tiropramide on intestine, particularly its pharmacodynamic and pharmacokinetic properties.

TITLE: The Effect of Desmopressin on the Amount of Bleeding in Patients Undergoing Coronary Artery Bypass Graft Surgery with a Cardiopulmonary Bypass Pump After Taking Anti-Platelet Medicine ABSTRACT.BACKGROUND: Coronary artery bypass grafting (CABG) is a common surgical intervention at the end-stages of coronary artery occlusion disease. Despite the effectiveness of CABG, it may have particular complications, such as bleeding during and after surgery. So far, there have been many drugs used to reduce bleeding. ABSTRACT.OBJECTIVES: This study aimed at investigating the effects of desmopressin on the amount of bleeding in patients undergoing CABG with a cardiopulmonary bypass pump (CPBP) who were taking anti-platelet medicine. ABSTRACT.METHODS: One hundred patients scheduled for elective CABG with a CPBP were included in a prospective, placebo-controlled, double-blinded clinical trial study. They were randomly divided into two groups. One group received desmopressin (40 μg) and the other group received a placebo (isotonic saline). Seven patients were excluded from the study, and 47 and 46 patients participated in the desmopressin and control groups, respectively. The methods of monitoring and the anesthetic techniques were similar in both groups, and all surgeries were performed by one surgeon. Variables including age, gender, pump time, aortic clamp time, duration of surgery, complications (e.g., nausea and vomiting, blood pressure changes), the necessity to receive blood products, and coagulation tests (prothrombin time, partial thromboplastin time, international normalized ratio, and bleeding time) were assessed. Data were statistically analyzed with SPSS software version 17. ABSTRACT.RESULTS: There was no significant difference between the groups regarding age, gender, pump time, clamp time, duration of surgery, complications, and the changes in hemoglobin and coagulation test measurements (P > 0.05). No significant difference was noted between the groups regarding the rate of bleeding after surgery (359.3 ± 266.2 in group D vs. 406.3 ± 341.6 in group P (control group); P = 0.208). However, the platelet changes after surgery in both groups were significantly different. The analysis revealed that the rate of thrombocytopenia after surgery was higher in the control group (P = 0.012). ABSTRACT.CONCLUSIONS: Our study showed that desmopressin could not reduce the amount of blood loss after CABG. Also, desmopressin did not have a significant effect on coagulation status. Therefore, based on the results of our study, it seems that the use of this medication cannot be a helpful for patients with any indication for CABG. BODY.1. BACKGROUND: Coronary artery disease (CAD) is one of the leading causes of death worldwide. Patients with CAD require coronary artery revascularization as well as other medical treatments (1). Coronary artery bypass grafting (CABG) is a common interventional surgery in order to treat end-stage coronary artery occlusion disease. Although CABG is an effective method, it may also come with particular complications during and after the surgery (1, 2). CABG may lead to disorders in platelet count, coagulation, and fibrinolysis. Excessive amounts of bleeding after surgery is a common problem that sometimes requires blood transfusions or even reoperation (3-6). Desmopressin is an artificial form of vasopressin which is commonly used to prevent bleeding in hemophilia and Von Willebrand disease (7, 8). Previous investigations have shown that desmopressin can decrease the amount of blood products needed in transfusion for cardiac surgery (9). However, some studies have indicated that desmopressin was not effective when used as a prophylaxis (10, 11). Still, some other studies have shown that using desmopressin could reduce bleeding in post-CABG patients, patients with long cardiopulmonary bypass pump (CPBP) times, and high-risk patients who had taken anti-platelet medication (9, 12-16). BODY.2. OBJECTIVES: According to the general importance of the issue and the conflicting results on the effects of desmopressin on bleeding after cardiac surgery, and also concerning the lack of previous studies which have assessed the effects of intranasal desmopressin on bleeding after cardiac surgery, we aimed to investigate the effects of desmopressin on the bleeding rates in patients who had undergone CABG with a CPBP after taking anti-platelet medicine. BODY.3. METHODS: We conducted a randomized double-blind clinical trial in the anesthesiology research center of Guilan University of Medical Sciences (GUMS) in Rasht, Iran. This study was approved by the ethics committee of GUMS with the reference number of 1930252810 and registered in the Iranian registry of clinical trials (IRCT) under the number IRCT201409304345N3. Inclusion criteria were Class II-III status according to the American society of anesthesiologists' physical status classification system, 3-vessel coronary artery disease, ejection fraction of ≥ 40%, reception of anti-platelet medicines including aspirin of 80 mg/day and clopidogrel of 75 mg/day before surgery, no history of coagulopathy or renal impairment, and having been scheduled for CABG with a CPBP. Exclusion criteria were a pump time > 120 minutes, concomitant valve procedure and CABG, inotrope administration for separating the patient from the cardiopulmonary pump, need for anticoagulant therapy during and/or in the post-operative period, and coronary endarterectomy. The sample size was determined according to the following formula derived from the study by Gratz et al. (17): α = 0.05 Z1-α/2 = 1.96 (95% confidence interval was noted) β = 0.20 Z1-β = 0.85 μ1 = 1176, S1 = 674 μ2 = 833, S2 = 311 Equation 1.n=(Z1-α2+Z1 - β)2(S12+S22)(μ1 -μ2)2 n = 50 Patients in each group. Using the method of random blocking, 100 patients were divided into two groups (desmopressin: group D and placebo: group P) with 50 patients in each group. At the time of hospitalization, the type of surgery and anesthesia method were explained for patients, and written informed consent was obtained. In the preoperative period, blood levels of hemoglobin (Hb), platelets, prothrombin time (PT), partial thromboplastin time (PTT), international normalized ratio (INR), and fibrinogen were measured and recorded. The anesthesiologist advised patients to fast the night before surgery. For premedication, oral lorazepam of 1 mg was administered the night before and one hour prior to surgery. An hour before induction of anesthesia, intramuscular morphine sulfate of 0.1 mg/kg was injected into all patients. Thirty minutes before anesthesia, the desmopressin or placebo was administered. In group D, two puffs of intranasal desmopressin (Sina Vista Daroo manufacturing, Tehran, Iran) were sprayed. Each puff contained 10 μg of desmopressin. In group P, 2 puffs of intranasal saline (normal saline in a desmopressin container) were sprayed. The patients and the anesthesiologist had no awareness about the drug type administered during and after surgery. Also, the laboratory technician was unaware of the drug type. The researcher was the only one who was aware of the medication so that he could take any necessary actions if complications occurred. Patients were monitored under standard protocols including 7-lead electrocardiography (EKG), pulse oximetry, and bispectral indexing (BIS) in the operating room. Before induction of anesthesia, the cardiac anesthesiologist inserted one peripheral venous catheter, a left radial artery catheter, and a central venous catheter under local anesthesia with lidocaine of 1%. Patients received 5 - 7 ml/kg isotonic saline before induction of anesthesia. After preoxygenation, anesthesia was induced by slow injection of midazolame of 0.05 mg/kg and sufentanil of 1 μg/kg (over the course of 10 minutes) intravenously, followed by infusion of 0.2 mg/kg cisatracurium for muscle relaxation. After intubation, anesthesia was maintained by propofol of 50 - 75 μg/kg/min, sufentanil of 0.2 μg/kg/h, and atracurium of 0.6 μg/kg/h. Then, a nasopharyngeal thermometer was inserted, and in order to maintain the depth of anesthesia, BIS during surgery was maintained between 40 and 60. Then, patients underwent median sternotomy, a standard technique was used to establish a CPBP. Activated clotting time (ACT) was also measured following the administration of 300 μg/kg heparin. Cardiopulmonary bypass was initiated if ACT ≥ 480 seconds. Upon completion of the grafts, if vital signs were stable, patients were separated from the pump, and the circulating heparin was antagonized by protamine sulfate. After the surgery, patients were transferred to the intensive care unit (ICU), resuming ventilation under close observation. Patients were extubated about six to eight hours after operation if they were awake, calm, had normal arterial blood gas analysis, and acceptable breathing status. If the patients had VAS > 3, intravenous morphine of 0.1 mg/kg was injected. Six hours after transferring the patients to the ICU, in group D, two puffs of intranasal desmopressin equivalent to 20 μg, and in group P, two puffs of intranasal placebo (isotonic saline), were sprayed. If the patients had nausea and vomiting (desmopressin complications), metoclopramide of 0.1 mg/kg was injected. In the ICU, the amount of blood in the chest bottle, Hb level, and platelet count were observed. If the Hb level reduced to less than 10gr/dL, or if the platelet level was less than 100,000/mm3, packed red blood cells (PRBC) or platelet concentrate were injected, respectively. Investigators measured the levels of Hb, platelets, fibrinogen, PT, PTT, and BT 24 hours after surgery. BODY.3. METHODS.3.1. STATISTICAL ANALYSIS: After collection, data were entered into SPSS software version 17. Data were reported by descriptive statistics (number, percent, mean, and standard deviation) and analyzed with a chi-squared test, an independent T-test, a Mann-Whitney U test, and a paired T-test. For intragroup comparison of variables after surgery, ANCOVA was used. A P value < 0.05 was considered as statistically significant, and a 95% confidence interval was noted. BODY.4. RESULTS: Of the 100 patients initially enrolled in this study, seven patients were excluded. Therefore, 47 and 46 patients participated in groups D and P, respectively. Two patients in group D and two patients in group P were excluded as a result of bleeding and reoperation required to control it. A prolonged cardiopulmonary pump time resulted in the exclusion of one patient in group D and two patients in group P (Figure 1). Figure 1.Enrollment and Outcomes There were no significant differences between the groups regarding age, gender, body mass index (BMI), ejection fraction (Table 1), duration of surgery, pump time, and aortic clamp time (Table 2). Also, there was no significant difference between the groups regarding the outbreak of complications such as nausea, vomiting, blood pressure alteration, and the amount of bleeding (P > 0.05). Table 1. Demographic Characteristics of the Patients Variables Group D Group P P Value Age, y 54.1 ± 7.58 56.92 ± 7.33 0.074 a Height, cm 166.3 ± 9.48 167.46 ± 3.04 0.215 a Weight, kg 71.8 ±7.63 72.4 ± 5.3 0.65 a BMI 25.93 ± 2.29 25.82 ± 1.66 0.79 a Ejection fraction (EF) 47.19 ± 4.18 47.86 ± 4.81 0.47 a Gender 0.217 b Male 35 (74.5%) 39 (84.8%) Female 12 (25.5%) 7 (15.2%) a Independent T-test. b Chi-squared test. Table 2. The Surgery Times, Pump Times, and Clamp Times of the Patients Variables Group D Group P P Value a Duration of surgery, h 2.89 ± 0.42 2.92 ± 0.44 0.73 Pump time, min 57.2 ± 9.01 56.7 ± 8.83 0.79 Aortic clamp time, min 37.34 ± 9.43 36.08 ± 9 0.51 a Independent T-test. Thirty-nine patients received PRBC, and 13 patients received platelet concentrate. The results showed no significant difference between these groups (p=0.472) (Table 3). Table 3. Frequency of Complications in the Two Groups Variables Group D Group P P Value Hypotension 0.533 a Yes 6 (12.8%) 8 (17.4%) No 41 (87.2%) 38 (82.6%) Nausea 0.109 a Yes 11 (23.4%) 5 (10.9%) No 36 (76.6%) 41 (89.1%) Vomiting 0.57 a Yes 2 (4.3%) 1 (2.2%) No 45 (95.7%) 45 (97.8%) Bleeding volume, mL 359.3 ± 266.2 406.3 ± 341.6 0.208 b Blood transfusion, packed cell 0.472 a Yes 18 (38.3%) 21 (45.7%) No 29 (61.7%) 25 (54.3%) Platelet transfusion 0.797 a Yes 7 (14.9%) 6 (13%) No 40 (85.1%) 40 (87%) Packed red blood cell transfusion, U 0.65 ± 1.06 0.82 ± 1.32 0.596 b a Chi-squared test. b Mann-Whitney U test. Table 4. Changes in Hemoglobin, Platelet, and Coagulation Results Before and After Surgery in the Two Groups Variables Group D Group P Estimate Within Group , P Value a Estimates Between Groups b Hemoglobin 0.134 Preoperative 13.63 ± 1.78 13.12 ± 2.18 0.221 Postoperative 11.71 ± 1.45 11.15 ± 1.31 0.058 Estimate within group, value c 0.0001 0.0001 Platelets 0.012 Preoperative 243489 ± 74577 240239 ± 46991 0.802 Postoperative 226176 ± 91502 194456 ± 46514 0.038 Estimate within group, P value c 0.042 0.0001 INR 0.665 Preoperative 1.0 ± 0.21 1.05 ± 0.14 0.86 Postoperative 1.11 ± 0.19 1.14 ± 0.22 0.964 Estimate within group, P value c 0.005 0.011 PT 0.463 Preoperative 12.85 ± 0.71 12.82 ± 0.62 0.88 Postoperative 13.28 ± 1.29 13.55 ± 2.12 0.941 Estimate within group, P value c 0.017 0.011 PTT 0.588 Preoperative 30.97 ± 5.96 30.98 ± 7.47 0.134 Postoperative 29.73 ± 8.63 28.77 ± 8.55 0.517 Estimate within group, P value c 0.029 0.085 BT 0.129 Preoperative 1.3 ± 0.39 1.42 ± 0.42 0.223 Postoperative 1.69 ± 0.56 1.55 ± 0.58 0.173 Estimate within group, P valuec 0.0001 0.285 Fibrinogen 0.265 Preoperative 302.9 ± 34.3 288.7 ± 45.6 0.091 Postoperative 261.4 ± 37.4 247.8 ± 35.1 0.075 Estimate within group, P value c 0.0001 0.0001 Abbreviation: BT, bleeding time; INR, international normalized ratio; PT, prothrombin time; PTT, partial thromboplastin time. a Independent T-test. b ANCOVA. c Paired T-test. A paired T-test revealed that the Hb levels decreased significantly in each group after surgery (P = 0.0001), but the difference was not statistically significant between the groups (0.134). The alterations of platelets in the D and P groups were statistically significant (P = 0.042, P = 0.0001, respectively). A T-test showed no significant difference in mean platelet levels between the groups before surgery (P = 0.802), but there was a significant difference between the groups in terms of the mean platelet levels after surgery (P = 0.038). The PT, PTT, INR, BT, and fibrinogen changes before and after surgery showed no significant differences between the groups (P > 0.05). BODY.5. DISCUSSION: Severe bleeding after cardiac surgery consistently remains a challenge for cardiac surgeons and anesthesiologists. Using a cardiopulmonary pump in cardiac surgery may induce blood coagulation disorders and platelet dysfunction, requiring transfusion of proper functioning platelets or the administration of other medicines (3-6). Up until now, diverse products including desmopressin, albumin, recombinant factor VII, and tranexamic acid have been used to reduce bleeding after cardiac surgery (12, 15, 18-20). Desmopressin is an artificial form of vasopressin or an antiduretic hormone (ADH) analogue that exerts its anti-hemorrhagic effects through increased plasma levels of factor VΙΙΙ and increased activity of the Von Willebrand factor. It also has a direct effect on the blood vessel. Our study showed no significant differences between the groups regarding the demographic characteristics, pump times, clamp times, and durations of surgery. Also, the frequency of complications such as nausea, vomiting, and hypotension was not significantly different between the groups. Furthermore, no significant difference was noted between the groups regarding the amount of blood collected in the chest bottle and the number of transfused PRBC units. Pleym et al. observed no significant difference in the amount of blood loss after prescribing 0.3 mcg/kg of desmopressin in patients undergoing CABG (21). They reported 606 and 601 cc of blood loss in the desmopressin and saline groups, respectively. They finally declared that taking desmopressin had no significant effect on bleeding after CABG in patients treated with aspirin the day before surgery. Their finding was consistent with our study, but with increasing doses of desmopressin, different results might be achieved. Cattaneo et al. showed that although the use of desmopressin reduced postoperative bleeding, it did not reduce the need for blood transfusions (12). In both groups, the same amount of blood was transfused. Also, in this study, researchers noted that the effectiveness of desmopressin depended on the amount of bleeding, and by an increase in bleeding of about 1.1 liters, the effect of desmopressin on reducing blood loss could be more apparent (12). It seems that low rates of bleeding could indicate the supposed ineffectiveness of desmopressin in our study (359.3 ± 266.2 mL in group D, and 406.3 ± 341.6 in Group P). In our study, it was observed that Hb levels dropped after surgery in each group, and this amount was significant. However, comparing Hb levels before and after surgery in both groups indicated no significant difference. Also, Pleym et al. noted similar results (21). Furthermore, platelet counts after surgery decreased significantly in both groups in our study. Platelet counts in group P were lower than those of group D, but this difference was not clinically considerable, and in both groups, postoperative platelet counts were enough to maintain homeostasis. In contrast to our study, Pleym et al. reported a different result (21). They indicated that despite a decrease in platelets after surgery in both groups, the rate of thrombocytopenia was higher in the desmopressin group. This different result might be due to other factors such as the cardiopulmonary pump effect. In addition, there was no significant difference between groups in BT before and after surgery (P = 0.129). In a study by Keyl et al. which examined the effects of aspirin and intravenous desmopressin of 0.4 μg/kg on platelet function in patients undergoing CABG, they observed that desmopressin did not affect platelet activity (22). In our study, 40 μg of intranasal desmopressin in the form of four puffs was administered before (two puffs) and six hours (two puffs) after surgery. Intranasal desmopressin at high dosages may produce an elevation of blood pressure, which disappears with reduction in dosage. Therefore, this drug should be used with caution in patients with coronary artery insufficiency and/or hypertensive cardiovascular disease and heart failure. For this reason, in our study, the lower dosage of desmopressin was used. In Keyl et al.'s study, similar to ours, desmopressin did not improve platelet function. However, their study focused on platelet activity more than platelet count. In our study, platelet count and BT were also investigated. Our results showed that PT and INR increased after surgery, but this difference was not significant when comparing the two groups. The only variable with a downward trend in coagulation parameters was PTT. However, a significant difference in this variable was not observed between the groups. Generally, desmopressin did not improve platelet count or function, nor did it improve coagulation status. In a review study, Wademan and Galvin (23) examined the effects of desmopressin in reducing bleeding after heart surgery. They announced that desmopressin could not induce beneficial effects either before or after heart surgery. In their study, desmopressin improved the hemostatic status in those patients consuming aspirin for seven days prior surgery, having a pump time of more than 140 minutes, or having preoperative platelet dysfunction. Also, in a meta-analysis, Levi et al. (24) showed that desmopressin might cause a slight reduction in the amount of bleeding after heart surgery without any clinical improvements. BODY.5. DISCUSSION.5.1. LIMITATIONS: In this study, we investigated the platelet counts and BT. It is recommended that in future studies, further platelet function tests such as optical aggregometry, flow cytometry, or PFA-100 could be used. Also, it seems that measuring the Von Willebrand factor and factor VΙΙΙ levels before and after surgery could prove to be valuable. BODY.5. DISCUSSION.5.2. CONCLUSION: Our study showed that desmopressin could not reduce the amount of blood loss after CABG surgery. Also, desmopressin did not have a significant effect on coagulation status. Therefore, based on our study, it seems that the use of this medication cannot be considered as helpful in candidates for CABG surgery.

TITLE: School Age Effects of the Newborn Individualized Developmental Care and Assessment Program for Medically Low-Risk Preterm Infants: Preliminary Findings ABSTRACT.BACKGROUND:: By school-age, even low-risk moderately preterm-born children show more neuro-cognitive deficits, motor impairments, academic underachievement, behavioral problems, and poor social adaptation than full-term peers. ABSTRACT.AIM:: To evaluate the outcomes at school-age for moderately preterm-born children (29-33 weeks gestational age), appropriate in growth for gestational age (AGA) and medically at low-risk, randomized to Newborn Individualized Developmental Care and Assessment Program (NIDCAP) or standard care in the Newborn Intensive Care Unit. At school-age, the experimental (E) group will show better neuropsychological and neuro-electrophysiological function, as well as improved brain structure than the control (C) group. ABSTRACT.MATERIALS AND METHODS:: The original sample consisted of 30 moderately preterm-born infants (29 to 33 weeks), 23 (8C and 15E) of them were evaluated at 8 years of age, corrected-for-prematurity with neuropsychological, EEG spectral coherence, and diffusion tensor magnetic resonance imaging (DT-MRI) measures. ABSTRACT.RESULTS:: E-performed significantly better than C-group children on the Kaufman Assessment Battery for Children-Second Edition (KABC-II) and trended towards better scores on the Rey-Osterrieth Complex Figure Test. They also showed more mature frontal and parietal brain connectivities, and more mature fiber tracts involving the internal capsule and the cingulum. Neurobehavioral results in the newborn period successfully predicted neuropsychological functioning at 8 years corrected age. ABSTRACT.CONCLUSION:: Moderately preterm infants cared for with the NIDCAP intervention showed improved neuropsychological and neuro-electrophysiological function as well as improved brain structure at school-age. BODY.INTRODUCTION: Preterm compared to full-term children perform more poorly in working memory, planning, visual spatial organization, and mental flexibility,[12] and are over-represented among early intervention and special education service recipients.[3] Early brain-based differences[45] contribute to long-term disabilities. Poor executive function appears related to basal ganglia/cerebellar volume reduction and sub-cortical white matter circuit disruptions between frontal, striatal, and thalamic regions.[67] Cumulative effects of medical complications[8] are compounded by the Newborn Intensive Care Unit (NICU) experience (exposure to bright lights, heightened sound, frequent interventions), which alters brain development.[91011] The Newborn Individualized Developmental Care and Assessment Program (NIDCAP)[12] provides a system of NICU care and environmental structure that supports preterm infants' early brain development. Several randomized, controlled NIDCAP trials reported significant neurobehavioral and neuro-electrophysiological improvement for high-risk preterm infants.[1314151617181920] School-age follow-up studies[2122] showed continued significant neurodevelopmental improvement. The current study tests NIDCAP-effectiveness into school-age for medically low-risk, appropriate for gestational age (AGA), moderately preterm infants and evaluates prediction by newborn-period neurobehavioral measures of school-age neuropsychological performance. BODY.MATERIALS AND METHODS.STUDY DESIGN AND ETHICS: Children born preterm, who had been studied in-NICU during a randomized control trial[23] (control-C and experimental-E), were assessed in follow-up at 8 years (y) of age corrected-for-prematurity (CA). The study protocol was approved by the hospital's Institutional Review Board for Research with Human Subjects. All school-age assessment personnel (neuropsychology, interviews, EEG, and MRI) were kept blind to original subject group assignments. BODY.MATERIALS AND METHODS.SUBJECTS: The original sample[23] consisted of 30 study infants (14C; 16E), recruited from the 46-bed level-III NICU, with an inborn population at a large urban tertiary care center. Family selection criteria included: Maternal age >14 years; no major medical or psychiatric illness, chronic medication treatment, and/or history of substance abuse; telephone accessibility; and English-language facility. Infant criteria included: Gestational birth-age 28 weeks 4 days (d) to 33 weeks 3 days; 5-minute Apgar >7; at birth AGA (5th-95th percentile)[24] in weight and head circumference; normal initial cranial ultrasound (s), MRI, and/or electroencephalogram (EEG); <72 hours ventilator and/or vasopressor support; prenatal care; absence of congenital/chromosomal abnormalities, congenital/acquired infections, prenatal brain lesions, and seizures. Of the 30 subjects, 23 (8C; 15E) returned at school-age [Figure 1]. Figure 1Consort chart BODY.MATERIALS AND METHODS.SUMMARY OF NEWBORN INTERVENTION AND STUDY RESULTS: E-infants received NIDCAP[12] from NICU-admission to 2 weeks corrected age. C-group care was the study NICU's standard care. At 2 weeks corrected age, E-infants showed significantly better neurobehavioral functioning (Assessment of Preterm Infants' Behavior-APIB[25]), increased brain functionality (EEG) with increased frontal to occipital brain connectivities,[4] and improved brain structure (MRI) with more mature internal capsule and frontal white matter fiber tracts. The relationship among neurobehavior, EEG, and MRI was significant. Nine-months corrected age E-group neurodevelopmental functioning (Bayley Scales of Infant Development, Second Edition[26]) was significantly improved. BODY.MATERIALS AND METHODS.PRIMARY AND SECONDARY SCHOOL-AGE HYPOTHESES: Significant E-group-favoring effects were hypothesized for visual-spatial planning, executive function and working memory, spectral coherence increase between long-distance bi-hemispheric frontal and parietal brain system, and improved fiber tract development in internal capsule and optic radiations. Significant relationships were hypothesized among school-age neuropsychological function, spectral coherence, and diffusion-tensor magnetic-resonance-imaging, and between newborn neurobehavioral and school-age neuropsychological function. BODY.MATERIALS AND METHODS.SAMPLE DESCRIPTION: Newborn background information was compared between the children who returned for school-age follow-up and those who did not. Newborn background information was also compared between returning school-age C-and E-group children. School-age anthropometric, medical, and academic history indices were measured or obtained by parent interview.[27] Parent-IQ, reportedly correlating with child functioning,[28] was measured with the Kaufman Brief Intelligence Test, Second Edition (KBIT-2),[29] yielding a Verbal IQ, Non-Verbal IQ, and Mental Processing Composite (Mean-x: 100; standard deviation-SD: 15). Should parent-IQ, hypothesized to be comparable between groups, correlate with child-IQ, all outcome measures would be corrected for parent-IQ. BODY.MATERIALS AND METHODS.SCHOOL-AGE NEURODEVELOPMENTAL OUTCOMES.NEUROPSYCHOLOGICAL MEASURES: The small sample size necessitated limited neuropsychological assessment. An experienced neuropsychologist performed the Kaufman Assessment Battery for Children, Second Edition (KABC-II)[30] yielding a Mental Processing Composite Index (x=100; SD=15), four Scale Indexes, (Sequential, Simultaneous, Planning, Learning), and two language subscales (Expressive Vocabulary, Verbal Knowledge); the Woodcock-Johnson III Tests of Achievement (WJIII)[31] with two Standard Cluster Scores (x=100; SD=15) (Broad Reading-Letter/Word Identification, Reading Fluency, Passage Comprehension; Academic Skills-Letter/Word Identification, Calculation, Spelling); and the Rey-Osterrieth Complex Figure Test (Rey),[323334] Copy, Immediate Recall and Delayed (20 minutes) Recall conditions assessing gestalt integration, executive function, spatial planning, and memory. The Rey Developmental Scoring System[3536] yields per condition 3 mutually exclusive scores, Organization; Structural Elements Accuracy; and Incidental Elements Accuracy. BODY.MATERIALS AND METHODS.SCHOOL-AGE NEURODEVELOPMENTAL OUTCOMES.NEUROPHYSIOLOGICAL MEASURES: EEG and MRI studies were conducted within 1 week of neuropsychological testing. A pediatric EEG-technologist collected thirty-two-channel EEG at a 256 Hz sampling rate (with 1-50 Hz bandpass filtering with 60 Hz mains filter) for 12 minutes of Eyes Closed alert state EEG. Paroxysmal eye, muscle, and body movements were visually identified and excluded. Figure 2 shows standard EEG electrode names and positions [Figure 2]. Analysis used the Laplacian reference-electrode-free format, sensitive to underlying cortex and insensitive to deep/remote EEG sources.[37] Residual eye blink/movement artifacts were removed with source component techniques.[3839] (BESATM software package). Spectral analysis, including spectral coherence calculation,[40] was performed (NicoletTM software package). Two Hz/data point (16 points/32 Hz) spectral resolution for 32 channels yielded 7936 individual coherence variables. Remaining low amplitude, artifactual contributions were removed by multivariate regression analysis,[41] utilizing signals proportional to known artifact sources. Coherence variable number was reduced by using in-house-developed[42] principal components analysis software suited to factoring large asymmetrical matrices. Forty coherence factors, previously created on an independent age-comparable normative sample (n=219) and reflecting 48% of total coherence variance,[4344] were formed on the current school-age subjects utilizing the previous principal-components-analysis-generated rule. Given the sample size, the first 20 factors were utilized in the subsequent analyzes. Figure 2Standard EEG electrode names and positions. Head in vertex view, nose above, left ear to left. EEG electrodes: Z: Midline: FZ: Midline Frontal; CZ: Midline Central; PZ: Midline parietal; OZ: Midline occipital. Even numbers, right hemisphere locations; odd numbers, left hemisphere locations: Fp: Frontopolar; F: Frontal; C: Central; T: Temporal; P: Parietal; O: Occipital. The standard 19, 10-20 electrodes are shown as black circles. An additional subset of 17, 10-10 electrodes are shown as open circles BODY.MATERIALS AND METHODS.SCHOOL-AGE NEURODEVELOPMENTAL OUTCOMES.NEUROSTRUCTURAL MEASURES: Diffusion-tensor-MRI evaluated underlying brain structure by quantitative assessment of brain connectivity to delineate relevant white matter pathways and measure myelination and axon integrity parameters. Data were acquired at 3Tesla (Siemens Tim Trio, Siemens, Erlangen, Germany) with an MR imager using a 32-channel head coil. High spatial resolution echo-planar diffusion-weighted images were acquired (24 cm FOV, matrix 128 × 128, 2 mm thick contiguous slices). Geometric distortion from magnetic susceptibility differences was minimized with a short echo time (TE=78 ms) and parallel imaging (iPAT 2). Thirty b=1000 s/mm2 images were acquired at directions evenly spaced on the sphere along with 5 baseline (b=0) images. Diffusion tensors were reconstructed, and 5 major fiber pathways were identified with a previously validated automated procedure.[45] Summary diffusion scalar measures of mean diffusivity, axial diffusivity, radial diffusivity, and fractional anisotropy were averaged in a streamline-density-weighted fashion.[46] along 5 major pathways: Arcuate Fasciculus (connecting posterior brain areas with Broca's area involved in complex language processing[47]); Corpus Callosum (thick white nerve band deep within the brain connecting the two hemispheres, supporting their communication and activity coordination); Cingulum (tracts receiving inputs from thalamus and neocortex; projecting to the entorhinal cortex; integral to limbic system; involving emotion, learning, memory, and executive function); Internal Capsule (massive white matter layer, major route inter-connecting cerebral cortex with brainstem and spinal cord); and Optic Radiations (axons carrying visual information from lateral geniculate nucleus relay neurons of thalamus to visual cortex). Children were scanned unsedated, awake, watching a cartoon or movie. Broad language processing tracts (arcuate fasciculus)[48] and early-developing basic hemispheres-connecting structures were thought to be least affected by NIDCAP; cingulum, internal capsule, and optic radiations related to memory, executive function, and visual-motor processing were hypothesized to be improved for E-children. BODY.MATERIALS AND METHODS.DATA ANALYSIS: The Biomedical Data Package 2007TM (BMDP)[49] supported statistical analyzes. Continuous variables were submitted to univariate analysis of variance (ANOVA) (BMDP-7D).[50] In cases of unequal variance, the Browne-Forsythe test of variance (F*) was used. Categorical variables were submitted to Fisher's exact probability test (FET) for 2 × 2, and Pearson's Chi-square (χ2) test for all other multiple row by column arrays.[4951] Two-tailed values of P<0.05 were considered statistically significant. Sample sizes provided 80% power to detect large between-group-effects, generally effect sizes >1.0.[52] Analyzes included stepwise discriminant analysis (BMDP-7M) for the neuropsychological, electrophysiological, and neurostructural domains; Wilks' lambda[53] and jack-knifed[5455] classification for ascertainment of two-group classification success per domain and across domains; canonical correlation analysis (BMDP-6M) to explore relationships among the neuropsychological, electrophysiological, and neurostructural domains at school-age, and between newborn and school-age neurobehavioral domains. BODY.RESULTS.SAMPLE.NEWBORN BACKGROUND FOR SUBJECTS WITH VERSUS WITHOUT SCHOOL AGE FOLLOW-UP: The subjects who returned for school-age follow-up were sicker at birth than those who did not return [Table 1].[56575859] Moreover, the school-age E-group had significantly lower 5-minute Apgar scores than the C-group [Table 2].[56575859] This biased results against the E-group. Table 1 Anthropometric, medical, and demographic background variables, participating vs. lost to follow-up subjects* Table 2 Anthropometric, medical, and demographic background variables, children seen at follow-up, control vs. experimental group* BODY.RESULTS.SAMPLE.SCHOOL-AGE BACKGROUND INCLUDING PARENT IQ: C-and E-school-age groups were comparable in age-at-testing, parent-IQ,[29] and anthropometric, medical, and demographic characteristics. E-children's head circumferences were somewhat larger [Table 3]. Parent Mental Processing Composite and non-verbal IQ[29] correlated significantly with Child Simultaneous Processing[30] (r=0.4309, P=0.05; r=0.4231, P=0.05, respectively). Therefore, all outcome measures were residualized by partial correlation and multivariate regression analysis (BMDP-6M) for Parent IQ. Table 3 Anthropometric, medical, and demographic variables at time of evaluation* BODY.RESULTS.NEURODEVELOPMENTAL SCHOOL-AGE OUTCOME.NEUROPSYCHOLOGICAL RESULTS: All subjects (8C; 15E) completed neuropsychological testing. E-performed significantly better than the C-children on KABC-II Composite Index Simultaneous Processing and on subtest Rover; subtest Triangles showed a trend [Table 4]. Both subtests assess planning, decision-making, executive function, and visual-spatial processing. The groups performed comparably on the WJ-III Broad Reading and Academic Skill Clusters [Table 5], as well as on the 9 Rey scores. However, on the Rey, Basal Level and Organization (Immediate Recall), along with Incidental Accuracy (Delayed Recall), showed a trend towards favoring E-over C-children [Table 6], indicating that E-children showed somewhat better overall gestalt integration, visual-motor planning, visual gestalt and detail memory, and executive function. Figure 3 shows a C-and an E-child's sample drawings. The KABC-II Simultaneous Processing and Rey differences are reminiscent of the earlier school-age study results for high-risk preterms[22] and the poorer visual-spatial planning, executive and memory functions reported for preterms without intervention.[6061] Table 4 Kaufman assessment battery for children, second edition Table 5 Woodcock-Johnson III Table 6 Rey-Osterrieth complex figure test Figure 3Rey-Osterrieth complex figure. The figure represents sample drawings from 2 study children, 1 from the Control group, a 9 year 3 month old born at 31 w 1 d GA; and 1 from the Experimental group, a 8 year 4 month old born at 31 w 4 d GA. The conditions displayed are from left to right: Copy, Immediate Recall, and Delayed Recall Discriminant analysis (10 KABC-II, 7 WJ-III subtests, 9 Rey-measures) identified 3 KABC-II (Rover, Atlantis, and Triangles) and 2 Rey (Organization/Immediate and Delayed Recall) measures that showed significant C-from E-group differentiation [Table 7]. Misclassified were 2 C-and 3 E-subjects. Table 7 Discriminant function analysis of neuropsychological measures BODY.RESULTS.NEURODEVELOPMENTAL SCHOOL-AGE OUTCOME.NEUROPHYSIOLOGICAL RESULTS: All school-age subjects completed neurophysiological assessment. Coherence-Factor-15 showed significantly decreased E-over C-group connectivity (P=0.001) between the right medial posterior frontal and right occipital regions from 2-18 Hz. This suggests release of frontal-associative cortex from overly-restricted visual-motor integration, freeing it for higher level functions. Factor-13 showed a trend towards significant group difference (P=0.112) [Figure 4]. Factor-13 (8-12 Hz, i.e., alpha), a broad, long-distance, and interhemispheric set of connectivities, demonstrated strong E-over C-group enhancement of left frontal lobe (typically dominant in motor and sensory processing functions) connectivity with multiple distant regions, especially in the contra-lateral hemisphere, suggesting better information and processing flow to and from left frontal lobe, indicative of better visual-spatial and broad high level judgment, planning, and executive control functions. Figure 4EEG Spectral coherence factors at school age, Control (C) (n = 8), Experimental (E) (n = 15). Head shown in vertex view, nose above, left ear to left. EEG frequency and coherence electrodes shown above head. Arrow color illustrates experimental group coherence; green = decreased, red = increased Discriminant analysis identified 3 coherence factors [Figure 4] significantly differentiating C-from E-children [Table 8]. Jackknifed classification success utilizing the 3 factors showed 91.3% correct subject classification.[5455] The factors included Coherence-Factor-7, increased for the E-subjects, a long distance bi-hemispheric factor (6-20 Hz) connecting parietal-associative regions with bilateral prefrontal cortex, consistent with more mature prefrontal cortex connectivities underlying organization, planning and executive function; Factor-12, (20-30 Hz), involving, similar to Factor-13, E-group connectivity increase of left lateral-frontal regions to homologous, broader, right lateral-frontal and anterior-temporal regions, likely sub-serving working memory; and again Factor-15, as interpreted above. These factors misclassified only 2 C-subjects. Table 8 Discriminant function analysis of EEG coherence factors Overall, the successful group-discriminating factors highlighted two increased bi-hemispheric, connectivities from left frontal to broad temporal and parietal regions and one decreased connectivity, freeing up frontal system function, mirroring earlier results.[222362] NIDCAP, for this population, appears to have increased connectivities strongly supportive of broad executive and complex planning functions as well as of working memory. BODY.RESULTS.NEURODEVELOPMENTAL SCHOOL-AGE OUTCOME.BRAIN STRUCTURAL RESULTS: Twenty-one (7 C; 14 E) subjects completed MRI study. For internal capsule and optic radiation, as hypothesized, E-showed a significantly stronger trend towards lower diffusivity (mean and radial diffusivity) than C-children. Internal capsule, axial diffusivity was also lower. The cingulum also showed significantly lower E-than C-group mean and radial diffusivity and a trend towards lower axial diffusivity; and the arcuate fasciculus showed a trend towards lower mean and radial diffusivity [Table 9]. Higher E-than C-group fractional anisotropy was observed only at the trend level for the cingulum. Other structures differed in the direction favorable to the E-group. Marginal fractional anisotropy findings were possibly due to the small sample [Table 10 and Figure 5]. Table 9 Diffusion tensor magnetic resonance imaging, diffusivity measures (C=7; E=14) Table 10 Diffusion tensor magnetic resonance imaging factors for fractional anisotropy, (C=7; E=14) Figure 5Mean diffusivity in cortico-spinal tract (internal capsule) at 8 years. Control children, top row, experimental children, bottom row. Mean diffusivity rendered onto trajectories of the cortico-spinal tract, and color coded from red (low) to yellow (high). (Yellow and brighter orange: Higher measure of mean diffusivity; darker orange and red: Lower measure of mean diffusivity Discriminant function analysis accessing all 20 diffusion-tensor-MRI variables identified 3 measures, corpus callosum radial diffusivity, cingulum fractional anisotropy, and internal capsule radial diffusivity, which significantly differentiated C-from E-children [Table 11]. Two C-and 2 E-subjects were misclassified. Despite reduced sample, diffusion-tensor-MRI successfully differentiated the groups. Table 11 Discriminant function analysis of diffusion tensor magnetic resonance imaging measures BODY.RESULTS.NEURODEVELOPMENTAL SCHOOL-AGE OUTCOME.CLASSIFICATION SUCCESS UTILIZING ALL THREE NEURODEVELOPMENT DOMAINS: When examining the relative group classification power of the 3 neurodevelopmental domains, discriminant analysis identified 2 coherence factors (15, 12) and 3 neuropsychological variables including KABC-II Triangles (measuring planning, decision-making, executive function, and visual-spatial processing) and Rey Organization-Immediate and Delayed Recall (assessing gestalt integration, executive function, spatial planning, and memory) measures which significantly differentiated C-from E-children [Table 12]. Two C-and 2 E-subjects were misclassified. Classification success was highly significant despite small sample size. Thus, overall, the EEG measures were most successful in discriminating C-from E-children. Table 12 Discriminant function analysis of neuropsychological, EEG and diffusion tensor magnetic resonance imaging measures BODY.RESULTS.NEURODEVELOPMENTAL SCHOOL-AGE OUTCOME.RELATIONSHIP BETWEEN NEUROPSYCHOLOGICAL AND SPECTRAL COHERENCE MEASURES: Canonical correlation between the discriminant-analysis- identified neuropsychological measures (KABC-II Rover, Atlantis, Triangles; Rey Immediate/Delayed Recall Organization Scores) and spectral coherence factors (12, 7, 15) showed a significant relationship (Bartlett's test, χ2=38.01, df=15, P<0.0160). One canonical variable described the relationship. KABC-II subtest Atlantis (memory storage/retrieval of verbal information) and Rey Organization-Delayed Recall (long-term storage retrieval of visual-spatial content and executive function) as well as Coherence Factors-12 and-7 correlated highest with the canonical variable. Thus, better verbal and executive function, spatial organization, planning, and memory were associated with stronger broad bilateral frontal and parietal connectivities. BODY.RESULTS.NEURODEVELOPMENTAL SCHOOL-AGE OUTCOME.RELATIONSHIP BETWEEN SPECTRAL COHERENCE AND DIFFUSION-TENSOR-MRI MEASURES: Canonical correlation between the discriminant-analysis- identified Coherence Factors-7,-12, and-15 and the diffusion-tensor-MRI measures, (corpus callosum radial diffusivity, cingulum fractional anisotropy, and internal capsule radial diffusivity) was marginally significant (χ2=14.79, df=9, P=0.0968). One canonical variable described the relationship. Variables correlating significantly with the canonical variable included Coherence Factors-15 (positive) and-7 (negative), and cingulum fractional anisotropy (positive), and internal capsule radial diffusivity (negative). Thus, measures of integrated central parietal and bilateral frontal connectivities coupled with frontal system functioning unhampered by restrictive visual motor input were associated with better-developed cingulum and internal capsule fiber tracts. BODY.RESULTS.NEURODEVELOPMENTAL SCHOOL-AGE OUTCOME.RELATIONSHIPS BETWEEN NEWBORN AND SCHOOL-AGE NEUROBEHAVIORAL FUNCTION: Canonical correlation showed a significant relationship between the 8 newborn APIB/Prechtl factor scores[23] and 4 KABC-II[30] Scale Indexes (Sequential, Simultaneous, Planning, Learning) (χ2=46.19, df=32 P=0.0501). One canonical variable described the relationship. APIB/Prechtl-Factor-3 (reactivity and hypersensitivity) and KABC-II Simultaneous Scale Index correlated significantly with the canonical variable. Newborns, who were less hypersensitive and over-reactive, had better KABC-II Simultaneous Scale scores at school age. Canonical correlation also identified a highly significant relationship (χ2 = 137.64, df=96, P=0.0034) between the APIB/Prechtl factor scores[23] and 12 school-age Rey[32] measures. One canonical variable described the relationship. APIB/Prechtl-Factors-2 (broad motor organization) and-3 again, (see above) correlated significantly with the canonical variable as did Rey Organization-Copy and Basal Level-Immediate Recall. Thus, the better organized motorically and less hyper-reactive/hypersensitive the newborn, the better the school-age child's overall Rey-production in Copy and Immediate Recall. The relationship between newborn neurobehavioral and 8-year neuropsychological function was strong and internally consistent. BODY.DISCUSSION: Results reported, consistent across the 3 domains tested, support the hypothesis that NIDCAP enhances low-risk AGA moderately preterm infants' long-term neurodevelopment, specifically complex planning, executive function, memory, and simultaneous non-verbal mental processing. E-children showed significantly better neurobehavioral functioning at 2 weeks corrected age[23] and better simultaneous processing and complex planning, memory, and executive function at school-age. Similarly, E-children at 2 weeks corrected age showed a pattern of increased long-distance connectivities between occipital and frontal regions,[23] and at school-age increased bilateral and across-midline broad frontal and parietal connectivities and release from overly connected visual-motor function. The NIDCAP experience supports better-differentiated brain connectivity development. Better developed connectivities between frontal systems and the parietal systems appears more conducive to better mental control, executive and memory functions.[63] This is reflected in Factor-15, supportive of frontal associative cortical functions, and Factor-13, supportive of mental processing, memory, executive function, attention to spatial relationships/location, responsivity to object shape, size, and orientation, and visual spatial working memory.[6465] The school-age neurostructural findings corroborated the neuropsychological and neurophysiological findings. Diffusion-tensor-MRI, which at 2 weeks corrected age showed improved E-group right and left internal capsule and frontal white matter tracts[23] showed at school-age improved internal capsule, cingulum, optic radiation, and arcuate fasciculus fiber tracts. This is the first report of school-age NIDCAP-effectiveness for low-risk AGA moderately preterm infants in terms of neuropsychological, electrophysiological, and brain-structural development. Results are internally consistent. NIDCAP is directed towards reliable reduction in stressful experiences and consistent return to base and restfulness to assure the infants' opportunities for continued behavioral re-integration of experiences, the foundation for increasingly well-differentiated modulation of function and the growth of well-differentiated brain-connectivities. Not all children returned for follow-up testing. It appears that the children who were more compromised at birth with lower Apgar scores at 5 minutes and higher SNAPPE-II, a newborn illness severity and mortality risk score, returned for school-age assessment. Moreover, the returning experimental group children were differentially sicker (lower Apgar scores at 5 minutes) in the newborn period than the returning control children. This emphasizes even more the effectiveness of the in-NICU NIDCAP intervention in improving neurodevelopmental outcomes at school-age. Interpretation of findings, nevertheless, requires caution. The study's most serious limitation is the small sample size. Substantiation by larger, longitudinal school-age follow-up studies is necessary to corroborate the result presented. Advances in newborn intensive care since the time of study also may have implications for result interpretation. The mechanisms underlying NIDCAP effectiveness remain to be discovered. The cost-effectiveness of NIDCAP, as compared to other in-NICU interventions,[66] remains to be evaluated. The long-term goal of the research is the wider dissemination of the NIDCAP approach. Given the encouraging findings, preterm infants and their families benefit when those responsible for NICU care are knowledgeable and well-educated in early brain development and provide opportunities for individualized developmental care. The highly dependent, sensitive, and rapidly developing preterm infants and their hopeful and vulnerable parents have little choice but to fully trust NICU staff. Professionals and NICU systems must live up to and warrant this trust.

TITLE: Management of ABSTRACT.INTRODUCTION:: Prevalence of diabetes and its complications have been a burden to the society from the ancient times, in the present and also will be in the future unless proper measures are taken to prevent its manifestation. There have been an increasing number of death associated-amputation cases which are mainly caused by nonhealing wounds. These facts urge researchers to develop new, more effective wound treatments for diabetic patients. ABSTRACT.AIM:: To evaluate and compare the effect of Katupila Kalka (Securinega leucopyrus [Willd.] Muell. leaf paste) and Tila Taila (Sesamum indicum oil) in Madhumehajanya Vrana (diabetic wounds/ulcers) with Betadine ointment. ABSTRACT.MATERIALS AND METHODS:: A total of 23 patients of Madhumehajanya Vrana were chosen and randomly divided into two groups (Group A and B). Patients of Group A were treated with local application of Katupila Kalka with Tila Taila, whereas, in Group B, Betadine ointment was applied on the affected parts, once a day in the morning for 30 days. The relief in signs and symptoms were assessed on scoring pattern. ABSTRACT.RESULTS:: In Group A, diabetic wounds treated with Katupila paste got healed within 28 days with minimal scar formation without any complications, whereas in Group B, wound was healed completely only in two patients within 28 days. In both the groups, no patients reported any adverse drug reaction during the entire course of treatment as well as in follow-up period. ABSTRACT.CONCLUSION:: Study concluded that the drug Katupila Kalka possesses Vrana Ropana (wound healing) activity with fine scaring. BODY.INTRODUCTION: In 2011, it was estimated that 366 million people worldwide had diabetes, representing roughly 8.3% of the adult population (20–79 years of age group). The number is expected to reach 552 million by 2030, representing 9.9% of the adult population. Diabetes is associated with various short and long-term complications, many of which, if left untreated, can be fatal. All these also have the potential to reduce the quality of life of people with diabetes and their families. The most common long-term complications affect the heart, eyes, kidneys, and feet.[1] Foot infections in persons with diabetes are a common and complex.[23] In addition to causing severe morbidities, they now account for the largest number of diabetes-related hospital bed-days[4] and are the most common proximate, nontraumatic cause of amputations.[56] Diabetic foot infections require careful attention and coordinated management, preferably by a multidisciplinary foot-care team.[7] Ayurveda has its own principles, but it is incorporating new theories and drugs in it and is presenting them according to its principles. Number of drugs known to Ayurveda since Vedic era is increasing with the time. Acharya Charaka says that a physician can add new drugs to these Mahakashayas by his own intelligence that will help him to treat the patients.[8] He also states that one should get the knowledge about plants from tribal people or cowherds as they know and use the plants in a better way.[9] However, after Acharya Bhavamishra, the process of addition of new drugs to science has diminished or somewhat ceased. It is said that the wounds/ulcers of the diabetic patient are cured with difficulty.[10] Even with the latest technology and modern medicine in hand, highly trained medical team around, yet the majority of the diabetic ulcers end up with more or less amputation of the concerned major or minor part of the lower limb. Securinega leucopyrus (Willd.) Muell. is known as Humari in India, Katupila in Sri Lanka and Spinous Fluggea in English. It is a desert climatic plant used topically in paste form for the healing of chronic and nonhealing wounds. Hence, in the light of the above references from classics, Katupila Kalka (paste) was selected for this study with an aim to assess its effect in the management of Madhumehajanya Vrana (diabetic ulcer). BODY.MATERIALS AND METHODS: Diagnosed patients of diabetic wounds were selected from outpatient and inpatient departments of Shalya Tantra irrespective of the age, sex, religion, caste, occupation, etc. The study was started after obtaining approval from the Institutional Ethics Committee (PGT/7-A/2012-2013/1964 dated 21/09/2012) and study has been registered under Clinical Trial Registry of India (No.: CTRI/2013/09/004020). Informed written consent from each patient was obtained before starting treatment course. BODY.MATERIALS AND METHODS.INCLUSION CRITERIA: Diagnosed cases of Madhumehajanya Vrana with Type I and Type II diabetes mellitus of 18–70 years age of either sex. BODY.MATERIALS AND METHODS.EXCLUSION CRITERIA: The patients with a history of tuberculosis, wounds other than diabetic wounds, uncontrolled hypertension, cardiac diseases, such as myocardial infarction, coronary artery disease, and ischemic heart disease were excluded in this study. Patients with human immune virus (HIV), venereal disease research laboratory (VDRL) and hepatitis-B test positive were also excluded from the study. BODY.MATERIALS AND METHODS.LABORATORY INVESTIGATIONS: Routine hematological parameters such as hemoglobin percentage, total leukocyte count, differential leucocyte count, bleeding time, clotting time, erythrocyte sedimentation rate; blood biochemical parameters such as fasting blood sugar, postprandial blood sugar, hepatitis B surface antigen, VDRL, and HIV; urine examination (routine and microscopic) was done to find out any other pathological condition. BODY.MATERIALS AND METHODS.RADIOLOGICAL EXAMINATION: X-ray of the affected foot (dorsoventral and lateral view) was done to rule out the osteomylitis of bone. BODY.MATERIALS AND METHODS.GROUPING AND POSOLOGY: Totally 23 patients of the diabetic wound were allocated randomly into two groups (Group A and B). Group A (n = 13): In this group, Katupila Kalka (paste of S. leucopyrus leaves) with Tila Taila (sesame oil) was applied locally on the affected part. The dressing was done once in the morning up to 1 month or healing of wound which is earlierGroup B (n = 10): In this group, patients were treated with dressing of Betadine ointment once daily in the morning and treated as the standard control group. The leaves of Katupila were obtained from Sri Lanka and authenticated[11] at Pharmacognosy Laboratory, IPGT and RA, Jamnagar. Patients were advised to continue their anti-diabetic and anti-hypertensive medications (Allopathic/Ayurvedic) during treatment. BODY.MATERIALS AND METHODS.ASSESSMENT OF THERAPY: The assessment of results was carried out mainly on the basis of relief in the signs and symptoms of the ulcers that are odor (Gandha), peri-wound skin (Varna), exudates level (Srava), type of exudates, pain (Vedana), wound measurement in cm (Akruti), and signs of infection [Table 1]. Table 1 Gradation for assessment of results Effect of therapy was statistically analysed by using student 't' test. BODY.MATERIALS AND METHODS.OVERALL EFFECT OF THERAPY: Cured: 100% improvement in subjective and objective parametersMarked improvement: 70–99%Moderate improvement: 50–69%Mild improvement: 30–49%Unchanged: <30%. BODY.OBSERVATIONS: The demographic data reveal that maximum patients were from the age group of 46 to 65 years (57%). Male patients were more that is 78.3% may be due to their irregular eating habits, addiction, excess standing work, and less care. Maximum patients were following Hindu religion (78%) and married (87%). Socioeconomically middle-class patients were in the majority (78.2%). The 78.27% of patients were having a history of diabetes and 21.73% of patients found with a history of both diabetes and hypertension. The 69.5% of patients had family history of diabetes. For the management of diabetic wound, 39.1% of patients had received medical treatment in past and 52.1% of patients had undergone surgery, whereas 8.6% of patients had both medical and surgical treatment. The majority of the patients that is 56.5% were suffering from wounds since 1 to 6 months, 34.7% of patients noted traumatic onset and maximum 73.9% were found with single wound. The 82.6% of patients were having Puya Srava (purulent exudates) and 95.6% of patients had Ghana Srava with 43.4% were having moderate Srava (discharge). Maximum numbers of patients presented without pain whereas only 34.9% of patients were found with pain. Bad odor was noted in all wounds and fever was found in maximum 73.91% of patients. In maximum 47.81% of patients the location of wound was at foot, as this part of the body more often gets trauma. BODY.RESULTS: In this study, infection was controlled within 3–4 days and foul smell was controlled after 3 days in patients of Group A. In Group B, foul smell from ulcer continues until the wound was healed. The surrounded skin color became normal skin color within 7 days in maximum patients (76%) of Group A, whereas only 32% patients showed normal peri-wound skin color in Group B. In Group A, 55% of patients showed a complete cessation of discharge within 7 days whereas in Group B, exudation found reduced in 34% of patients at the end of 28th day. In all patients, pain sensation at wound site regained by 7th day in Group A, due to neo-vascularization and restored pain sensation in nerve fibers. In Group A, 12 patients wound size was reduced completely within 28 days, whereas in Group B, only 29% of patients wound size was reduced completely within 28 days. In Group A, signs of infections relieved 61% by the 7th day and totally relieved at the end of 28th day, whereas in Group B, patients infection was relieved 41% by the end of 28th day [Tables 2 and 3]. Table 2 Effect of therapy on signs and symptoms of wound in Group A Table 3 Effect of therapy on signs and symptoms of wound in Group B In Group A, 92.3% of patients were completely cured, whereas in Group B, 20% of patients were completely cured [Figure 1]. Wound healing effect of Katupila in four cases is depicted in Figure 2. Figure 1Overall effect of therapy Figure 2Before and after treatment wound status of Katupila treated cases BODY.DISCUSSION: In this study, foul smell was controlled after 3 days in Group A patients, which was found statistically significant (P < 0.001). In Group B, foul smell from ulcer continues until the wound was healed. The Krimighna, Vishaghna, Rakshoghna (antimicrobial) properties of the Katupila helped to control the local infection and ultimately the bad odor.[12] In Group A, 100% normal skin coloration was achieved which was found statistically significant (P < 0.001). In 32% of patients of Group B, peri-wound skin color became normal which was found statistically significant (P > 0.05). Katupila may have Rakta Shodhana, Pittashamana, Varnya, Twaka Prasadana properties which helped to improve the surrounding wound skin color. Complete cessation of discharge within 7 days was observed in 55% of patients of Group A, while in remaining patients discharge was reduced by 28th day which was statistically significant (P < 0.001). Krimighna property of drug helped to control the infection. Hence, it can be said that Katupila paste had a better role than Betadine to reduce exudates level. In Group A, 77% of patients presented without pain while the pain was reported in 23% of patients due to diabetic neuropathy. After treating with Katupila, pain sensation regained by 7th day due to neo-vascularization and restored pain sensation in nerve fibers. The statistically insignificant result in pain relief was seen due to the restoration of pain sensation by correcting neuropathy. The Snigdha Guna of Tila Taila helped in control the vitiation of Vata and helped to relief in pain.[13] In Group B, there was minimal neovascularization, but the pain was relieved in 25% of patients due to the healed wounds which were statistically significant (P > 0.05). Hence, it can be inferred that Katupila is better than Betadine ointment to regain pain sensation (initial stage) as well as to relieve pain (in later stage). Complete wound healing was observed within 28 days in 92% cases so wound size reduction was found statistically highly significant (P < 0.001) which may be due to Katupila which may have Lekhana (scraping), Shodhana (cleaning) and Ropana (healing) properties. Madhura Rasa, Prinana (nourishment), Dhatuvardhana (tissue strength) and Dhatuposhana (nutrition) actions of Katupila helped to reduce the wound size by promoting healing and the rate of contraction. The infection at the wound site was assessed on the basis of odor, exudate level, local temperature, and swelling in both the groups. In Group A, signs of infections relieved 61% by the 7th day and totally relieved at the end of 28th day. Local application of Katupila reduces the discharge, swelling, pain by controlling the bacterial load, so wound became Shuddha (clean or healthy). Whereas in Group B, infection was relieved in 41% by the end of 28th day. Both drugs have shown statistically highly significant (P < 0.001) result in controlling infection. However, on the comparison, the duration of infection control by Katupila paste (that is 1st week) was found earlier than Betadine which took longer duration in controlling infections. The strength and positive finding of this study is Katupila Kalka had the potential to cure the diabetic wounds early than Betadine ointment. Findings in previously published case reports also showed the good potential in wound healing by Katupila.[1415] None of the adverse drug reaction noted during the treatment period. A reverse pharmacological study in the suitable animal model is a further scope of this work. As this drug is being used by traditional people and of herbal origin, there is a need to generate safety and toxicity profile of Katupila. The limitation of this research work is less number of patients due to the limited time of the postgraduate study. BODY.CONCLUSION: The study concluded that Katupila Kalka (paste) possesses highly qualitative efficacy in "Vrana Ropana" with fine scaring. Therefore, it may be recommended that this low cost, easily preparable application may be prescribed for continuous use for the healing purposes. BODY.CONCLUSION.FINANCIAL SUPPORT AND SPONSORSHIP: IPGT & RA, Gujarat Ayurved University, Jamnagar. BODY.CONCLUSION.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: Physiological effects of invasive ventilation with neurally adjusted ventilatory assist (NAVA) in a crossover study ABSTRACT.BACKGROUND: Neurally Adjusted Ventilatory Assist (NAVA) is a mode of assisted mechanical ventilation that delivers inspiratory pressure proportionally to the electrical activity of the diaphragm. To date, no pediatric study has focused on the effects of NAVA on hemodynamic parameters. This physiologic study with a randomized cross-over design compared hemodynamic parameters when NAVA or conventional ventilation (CV) was applied. ABSTRACT.METHODS: After a baseline period, infants received NAVA and CV in a randomized order during two consecutive 30-min periods. During the last 10 min of each period, respiratory and hemodynamic parameters were collected. No changes in PEEP, FiO2, sedation or inotropic doses were allowed during these two periods. The challenge was to keep minute volumes constant, with no changes in blood CO2 levels and in pH that may affect the results. ABSTRACT.RESULTS: Six infants who had undergone cardiac surgery (mean age 7.8 ± 4.1 months) were studied after parental consent. Four of them had low central venous oxygen saturation (ScvO2 < 65 %). The ventilatory settings resulted in similar minute volumes (1.7 ± 0.4 vs. 1.6 ± 0.6 ml/kg, P = 0.67) and in similar tidal volumes respectively with NAVA and with CV. There were no statistically significant differences on blood pH levels between the two modes of ventilation (7.32 ± 0.02 vs. 7.32 ± 0.04, P = 0.34). Ventilation with NAVA delivered lower peak inspiratory pressures than with CV: -32.7 % (95 % CI: -48.2 to –17.1 %, P = 0.04). With regard to hemodynamics, systolic arterial pressures were higher using NAVA: +8.4 % (95 % CI: +3.3 to +13.6 %, P = 0.03). There were no statistically significant differences on cardiac index between the two modes of ventilation. However, all children with a low baseline ScvO2 (<65 %) tended to increase their cardiac index with NAVA compared to CV: 2.03 ± 0.30 vs. 1.91 ± 0.39 L/min.m2 (median ± interquartile, P = 0.07). ABSTRACT.CONCLUSIONS: This pilot study raises the hypothesis that NAVA could have beneficial effects on hemodynamics in children when compared to a conventional ventilatory mode that delivered identical PEEP and similar minute volumes. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01490710. Date of registration: December 7, 2011. BODY.BACKGROUND: To better match the level of ventilator assistance to the patient's needs, manufacturers have developed new modes that deliver a level of assistance proportional either to the patient's inspiratory muscle effort, with proportional assist ventilation (PAV); or to the diaphragmatic electrical activity (EAdi), with neurally adjusted ventilatory assist (NAVA) [1]. For now, only NAVA is usable for infants. With this mode of mechanical ventilation, the collected electrical signal allows synchronization of ventilation to spontaneous breathing efforts of the child, as well as permitting pressure assistance proportional to the electrical signal. NAVA provides both fine synchronization of respiratory support and pressure assistance varying with the needs of the child. To our knowledge, no data have been published on the impact of NAVA on the hemodynamics in children mechanically ventilated. The impact of mechanical ventilation on the circulatory system remains a concern in some infants following surgery for repair of congenital cardiac defects [2]. After the completion of the operation, when there is consensus regarding a good surgical result, many children nowadays are rapidly weaned and successfully separated from mechanical ventilation shortly after the procedure. However, duration of mechanical ventilation is inversely proportional to the patient's age and directly related to the duration of cardiopulmonary bypass and complexity of the surgical procedure [3]. This physiologic study with a randomized cross-over design compared hemodynamic parameters when neurally adjusted ventilatory assist (NAVA) or conventional ventilation (CV) was applied. BODY.METHODS: This study was performed in the 12-bed PICU of the university hospital center of Nantes (France), between June 2012 and March 2013. Last year, 712 children were admitted in this unit. Among them, 182 underwent a cardiac surgery with cardiopulmonary bypass (55 % were extubated within 8 h). This trial was registered (ClinicalTrials.gov Identifier: NCT01490710). Research has been performed in accordance with the Declaration of Helsinki and has been approved by an appropriate ethics committee (Comité de Protection des Personnes Ouest III de POITIERS, Réference comité: Protocole n°11.07.20). The parents of all the children gave their written consent. BODY.METHODS.STUDY DESIGN: PROSPECTIVE RANDOMIZED CROSS-OVER STUDY: After a baseline period, children received conventional ventilation (CV) and NAVA during two consecutive periods of 30 min, in random order. This randomization was performed by contacting a server to define the order in which the two modes of ventilation were administered. There was no washout time between these two periods. During the last 10 min of each period at a steady state, hemodynamic parameters, respiratory parameters, blood gas and cardiac index were collected. No changes in PEEP, FiO2, sedation or inotropic doses were allowed during these two periods. The study protocol is depicted in Fig. 1.Fig. 1Study design. Baseline measures were collected when patient met stability conditions. No changes in PEEP, FiO2, sedation nor inotropic doses were allowed during the two 30-min study periods. NAVA = Neurally Adjusted Ventilatory Assist; CV = Conventional Ventilation BODY.METHODS.PARTICIPANTS: Children hospitalized for postoperative care after cardiac surgery were considered for inclusion. Inclusion criteria were: admission weight >5 kg; invasive ventilatory support ongoing; NAVA ventilation available; sedation status allowing the NAVA ventilation functioning. Non inclusion criteria were: parental refusal; oesophageal disease prohibiting gastric tube; brain damage conflicting with spontaneous ventilation; clinical instability requiring treatment and/or management inconsistent with research. BODY.METHODS.INTERVENTION: Children were ventilated with a Servo-i ventilator (Maquet Critical Care, Solna, Sweden). During a baseline period, conventional ventilation was applied and adjusted according to blood gases. After the baseline period in which NAVA probe and Doppler monitoring were introduced, all children received conventional ventilation (CV) and NAVA during two consecutive periods of 30 min, in random order. The ventilation settings were supposed to provide the same minute volumes. BODY.METHODS.INTERVENTION.CONVENTIONAL VENTILATION (CV): With the goal of providing the lowest peak pressures, we used the Volume Control Intermittent Mandatory Ventilation (VC-IMV). Ventilatory parameters were as follows: tidal volume (between 5 and 8 ml/kg), respiratory rate (between 20 and 30 cycles/min). Pressure Support was set at 12 cmH2O above the PEEP. A flow pneumatic trigger was used to synchronize the support to spontaneous breaths. The flow pattern during the volume control ventilation was constant flow. The inspiratory time was initially set at 0.5 s and adjusted according to the inspiratory flow waveforms if needed. The respiratory rate of VC-IMV was set at a high level (between 20 and 30 cycles/min), not allowing many spontaneous cycles. The CV settings were adjusted during the baseline period with no changes allowed during the study period. BODY.METHODS.INTERVENTION.NEURALLY ADJUSTED VENTILATORY ASSIST (NAVA): During NAVA, the ventilator delivers a pressure proportional to the electrical activity of the diaphragm (EAdi). The settings in NAVA mode included PEEP, FiO2 and level of NAVA assistance. The NAVA level was set to 1 cmH2O/μV. We had previously observed that children ventilated with NAVA usually adjust their EAdi to maintain a normal blood pH by preserving physiologic tidal volumes and minute volumes. This is no longer true when children are highly sedated. Backup ventilation was Pressure Control set at 12 cmH2O above PEEP in case of failure EAdi signal detection. During NAVA, the positive pressure is triggered when the deflection in the EAdi curve exceeds 0.5 μV, and is cycled-off when the EAdi drops to 70 % of its peak value. BODY.METHODS.OUTCOMES MEASUREMENTS.RESPIRATORY MEASUREMENTS: Peak inspiratory pressures (PIP), PEEP, FiO2, Tidal volume (VT), Minute volume, EADI max, Respiratory rate, were recorded for all infants during the last 10 min of each period. Measurements of each parameter were repeated at steady state a total of six times to guarantee reliability and reproducibility. We collected the most representative values rather than gathering data at constant interval because of the variability of the respiratory measurements in NAVA. Hemodynamic and respiratory parameters were collected always in the same time. Respiratory system compliance (Crs) according to the ventilation mode was estimated by the ratio VT/(PIP-PEEP) in ml/kg.cmH2O. BODY.METHODS.OUTCOMES MEASUREMENTS.HEMODYNAMIC MEASUREMENTS: Cardiac index (CI, L/min.m2) was assessed by transesophageal Doppler ultrasonography (CardioQP, Gamida, France) [4]. Other hemodynamic parameters included heart rate, arterial pressures (by means of an arterial catheter placed before the surgery), and central venous pressure (by means of a central venous catheter inserted into the right internal jugular vein). Cerebral Near InfraRed Spectroscopy (NIRS) was measured with a non invasive optical monitor of regional cerebral oxygen saturation (Invos Oximeter, Covidien, Boulder, CO). The pulse oximeter perfusion index was provided by the monitors (Intellivue MP70 monitor, Philips Medical Systems) [5]. Hemodynamic parameters were collected always in the same time than respiratory parameters. Left cardiac work index [6] was calculated as: LCWI (kg.min/m2) = CI × MAP × 0.0136, with CI for cardiac index and MAP for mean arterial pressure. BODY.METHODS.OUTCOMES MEASUREMENTS.BIOLOGICAL MEASUREMENTS: At the end of each period (CV and NAVA), a blood sample was taken through the internal jugular catheter to measure pH, PCO2, and central venous oxygen saturation (ScvO2). These measurements were performed in the PICU by a blood gases analyser (GEM Premiere 4000, Instrumentation Laboratory UK Ltd). BODY.METHODS.STATISTICAL ANALYSIS: Each parameter collected in the last 10 min of each study period was averaged, and compared by a Wilcoxon test for paired samples. Measurements in NAVA and CV are reported as median (interquartile). Changes in parameters during NAVA compared to CV are expressed as mean percentage (95 % CI, P). The P-value taken to indicate significance was P < 0.05. For this pilot study no sample size was calculated. BODY.RESULTS: The study was concluded after one year. Technical problems were greater than expected: a measure of cardiac output sufficiently accurate, and especially stable in time, was very difficult to reach in children who already had a stomach tube for the NAVA ventilation. We asked the research office of our institution for continuing recruiting more patients. In view of the technical difficulties we had, this request was not accepted. Nine children were randomized. The quality of the surgical repair was always checked by a cardiologist prior to inclusion in the study. Analysis was performed on only six of them. We were unable to start NAVA on one child due to a lack of EAdi signal capture. Thereafter, when the NAVA signal was not stable enough, children were not included. In two other children we were unable to record a Doppler signal likely due to interference between the CardioQP probe and the EAdi probe both intra oesophageal placed. Patient characteristics are provided in Table 1. The mean (± SD) age was 7.8 ± 4.1 months, and weight was 6.7 ± 1.2 kg. Four children had low central venous saturation (ScvO2 < 65 %) at the baseline period: the children 1, 2, 3, 6 in the Table 1.Table 1Children characteristicsChild123456GenderMMMFFFAge (mo)313410611Weight (kg)5.78.97.06.05.87.0CardiopathyAVSDVSD with pulmonary stenosistetralogy of Fallotatrial septal defectcoarctation of the aortapulmonary atresia with VSDCardiopulmonary bypass duration (min)9683130480130Time between PICU entry and study (hours)20720457Baseline cardiac index (L/min.m2)2.002.851.932.602.751.85Milrinone (mcg/kg/min)0.90.50.30.500.5Adrenaline (mcg/kg/min)000000.05ScvO2 (%)646352826852Perfusion index (%)0.313.650.603.621.900.87Random orderCV then NAVANAVA then CVCV then NAVACV then NAVANAVA then CVNAVA then CVAll children were receiving morphine 0.5 mg per kilo per day. All, except the 2 first, were receiving midazolam 40 micrograms per kilo per hour AVSD atrioventricular septal defect, VSD ventricular septal defect, NAVA neurally adjusted ventilatory assist, CV conventional ventilation, ScvO 2: central venous oxygen saturation Respiratory parameters, hemodynamic parameters, and blood gases are provided in Table 2. The ventilatory settings resulted in similar minute volumes and in similar tidal volumes in NAVA compared to CV. These similar minute volumes in each periods of the study were supposed when the study was designed. Nevertheless, significant lower peak inspiratory pressures (PIP) were observed during NAVA compared to CV. Apparent respiratory system compliance improved with NAVA in all children. We observed higher systolic arterial pressures during NAVA compared to CV. Pulse oximeter perfusion index were also higher during NAVA.Table 2Respiratory parameters, hemodynamics and biological dataRespiratory parametersCVNAVANAVA versus CV P Respiratory rate (/min)30 (7)35 (13)+26.4 (-3.6, +56.4)0.17VT (ml/kg)6.6 (0.7)6.9 (0.3)+5.3 (-5.5, +16.1)0.46Minute volume (L/min)1.7 (0.4)1.6 (0.6)+5.4 (-4.3, +15.1)0.67PIP (cm H2O)21 (6)11 (4)-32.7 (-48.2, -17.1)0.04PEEP (cm H2O)4 (2)4 (2)-(a)EAdi max (microVolt)3.9 (3.8)6.3 (1.4)+61.2 (-4.6, +126.9)0.34SpO2 (%)98 (3)96 (3)-1.2 (-2.8, +0.5)0.14FiO2 30 (4)30 (4)-(a)Crs (ml/kg.cm H2O)0.37 (0.19)0.87 (0.32)+98.4 (+43.8, +153.0)0.04Hemodynamic parametersHeart rate156 (15)156 (22)+2.1 (-0.7, +4.5)0.17Systolic arterial pressure (mmHg)93 (6)99 (13)+8.4 (+3.3, +13.6)0.03Diastolic arterial pressure (mmHg)54 (12)57 (6)+3.6 (-3.0, +10.1)0.46Central venous pressure (mmHg)11 (5)10 (5)+3.9 (-5.3, +13.1)0.92Cerebral NIRS (%)62 (5)61 (3)+1.6 (-2.6, +5.8)0.34Cardiac index (L/min.m2)2.33 (0.84)2.26 (0.70)+1.4 (-3.4, +6.2)0.17Pulse oximeter perfusion index (%)1.50 (2.45)1.78 (2.29)+18.8 (+3.0, +34.7)0.04Venous blood gasespH7.32 (0.04)7.32 (0.02)-0.1 (-0.4, +0.2)0.34PCO2 (mm Hg)47.3 (5.1)45.8 (8.1)+0.6 (-5.1, +6.3)0.50ScvO2 (%)60.1 (20.9)58.4 (15.4)+3.3 (-5.7, +12.2)0.60Measurements in NAVA and CV are reported as median (interquartile). Variations between NAVA versus CV are reported as mean percentage (95 % CI). Statistical analyses between NAVA versus CV were performed by a Wilcoxon test for paired samples(a) No changes in PEEP, FiO2, neither in sedation or inotropic doses were allowed during these study periods NAVA Neurally adjusted Ventilatory Assist, CV Conventional Ventilation, V T tidal volume, PIP peak inspiratory pressure, Crs respiratory system compliance, EAdi electrical activity of the diaphragm, ScvO 2 central venous oxygen saturation Cardiac index during NAVA compared to CV did not statistically differ. However, all four children with ScvO2 < 65 % tended to have higher cardiac index after 30 min of ventilation with NAVA compared to CV: 2.03 ± 0.30 vs. 1.91 ± 0.39 L/min.m2 (median ± interquartile, P = 0.07). Individual values of cardiac index and ScvO2 of children with low baseline ScvO2 are shown in Fig. 2. Upper values of pulse oximeter perfusion index were also observed. Turning now to left cardiac work index, the increase is +12.4 % (95 % CI: +3.8 % to +20.9 %, P = 0.07).Fig. 2Cardiac index and ScvO2 of the low baseline ScvO2 infants, according to the ventilation mode. White bars are cardiac index when CV is applied; back bars are cardiac index when NAVA is applied. The triangles above the bars indicate the values of the corresponding ScvO2 (%). ScvO2 = central venous oxygen saturation; NAVA = Neurally adjusted Ventilatory Assist; CV = Conventional Ventilation BODY.DISCUSSION: NAVA can be used in infants receiving postoperative mechanical ventilation after congenital heart surgery, as previously reported [7–10]. Infants included in this study all weighed less than 10 kg and had been operated less than 24 h before. To deliver a same tidal volume, NAVA required lower inspiratory pressures than conventional ventilation and had some beneficial effects on hemodynamics. Several data validate that the children were not over assisted when ventilated in CV: the delivered volumes, EAdi max, PCO2 and pH are similar in CV compared to NAVA. We suggest that lower EAdi peaks observed in CV are related to the normalization of the blood pH by CV ventilation. If children were over-assisted in CV, EAdi would have been missing, and minute volume would have been high, with a blood alkalosis. In a large randomized controlled trial where NAVA was used as a primary mode of ventilation, lower peak inspiratory pressures were found in the NAVA group [11]. It looks as if gas volume delivery, and probably alveolar expansion, required less pressure. This could be enhanced by an improvement in patient–ventilator interactions with a greater respiratory variability as reported in previous studies [12–16]. The effectiveness of this mode of mechanical ventilation was associated with some beneficial effects on the hemodynamics. Systolic arterial pressures and pulse oximeter perfusion index were significantly higher in NAVA. This pulse oximeter perfusion index provides a monitoring of illness severity in neonates [5]. In fact, we observed that the children with a low cardiac index had also a low perfusion index. This index was significantly higher when the children were ventilated with NAVA compared to CV. The overall values of the cardiac index and of the central venous oxygen saturation during NAVA compared to CV were not significantly higher, but there are possibly no reasons to observe an increase in these two parameters when tissue perfusion is already effective. And actually, the four children with a low ScvO2 trend to have a higher cardiac index after 30 min of ventilation with NAVA (Fig. 2). Moreover, this increase in cardiac index is associated with higher systemic pressures and then higher left cardiac work index. Lower oxygen content in venous blood usually reflects an oxygen balance disrupted between oxygen supply and demand. Ventilation with NAVA could allow the body to increase the cardiac output to ensure adequate oxygen availability. These children had congenital heart disease affecting the right heart: VSD with pulmonary stenosis, tetralogy of Fallot, and pulmonary atresia with VSD. Positive pressure ventilation and PEEP often result in increased right ventricular afterload due to capillary compression [3, 17]. Lower intrathoracic pressures in NAVA than in conventional ventilation could improve the right ventricular function during inspiration by reducing the right ventricular afterload. An improvement in apparent respiratory system compliance was observed when NAVA is applied. Cyclic intrathoracic pressure changes, characteristic of spontaneous breathing, could be preserved. Both fine synchronization of respiratory support and pressure assistance varying with the needs and the spontaneous breathing of the child could improve the pulmonary compliance. Nonetheless, it is important to note that we did not record the transpulmonary pressure. It is likely that, while the airway pressure decreased during NAVA, an increase in esophageal pressure swings occurred because of the patient respiratory efforts. This could have participated in the decrease in apparent compliance improvement [18]. This study has some limitations. Firstly, few children were studied. Many children dropped out due to early extubation in the operation room or immediately on arrival in the intensive care unit. Technical problems were greater than expected: a measure of cardiac output sufficiently accurate, and especially stable in time, was very difficult to reach in children who already had a stomach tube for the NAVA ventilation. Nevertheless the cross-over design of this study has two advantages over both a parallel study and a non-crossover longitudinal study [19]. The influence of confounding covariates is reduced because each cross-over patient serves as his or her own control. Cross-over designs are also statistically efficient and require fewer subjects than do non-crossover designs (even other repeated measures designs). Secondly, the agreement of the cardiac output measurement between the transoesophageal Doppler probe using CardioQP and the thermodilution technique during heart catheterisation in paediatric patients was described as weak [20]. However the CardioQP seems to be capable of detecting slight changes in cardiac output for critically ill children [21, 22]. This bed-side device for cardiac output measurement is minimally invasive, and provides better results in monitoring the slight variations rather than in measuring absolute values. As we were performing a cross-over study, we were interested primarily by changes. Finally, only comparison with CV-SIMV with relatively high preset respiratory rate preventing many spontaneous cycles was made, not with other ventilation strategies. BODY.CONCLUSION: In conclusion, this cross-over study provides new data on the NAVA ventilation. This pilot study raises the hypothesis that a ventilatory assistance with NAVA could provide improvements in hemodynamics when compared to a conventional ventilatory mode that delivered identical PEEP and similar minute volumes. Thus, because minute volumes were not different between the two modes of ventilation, hemodynamic effects are linked to NAVA mode itself and NOT to incorrect settings in conventional ventilation. Further studies with larger population are needed to confirm these promising results.

TITLE: Gabapentin as an adjuvant for postoperative pain management in dogs undergoing mastectomy ABSTRACT: This study aimed to evaluate the analgesic efficacy of gabapentin as an adjuvant for postoperative pain management in dogs. Twenty dogs undergoing mastectomy were randomized to receive perioperative oral placebo or gabapentin (10 mg/kg). All dogs were premedicated with intramuscular acepromazine (0.03 mg/kg) and morphine (0.3 mg/ kg). Anesthesia was induced with propofol (4 mg/kg) intravenously and maintained with isoflurane. Intravenous meloxicam (0.2 mg/kg) was administered preoperatively. Postoperative analgesia was evaluated for 72 hr. Rescue analgesia was provided with intramuscular morphine (0.5 mg/kg). Dogs in the Placebo group received significantly more morphine doses than the Gabapentin group (P=0.021), despite no significant differences in pain scores. Perioperative gabapentin reduced the postoperative morphine requirements in dogs after mastectomy. BODY: Mammary tumors are the most common neoplasias in dogs and are commonly treated by an extensive mastectomy which results in inflammation, edema and moderate to severe postoperative pain [21]. Postoperative pain is not purely nociceptive in nature and may consist of inflammatory, neurogenic and visceral components [17]. Therefore, multimodal analgesic techniques utilizing a number of drugs which act on different analgesic mechanisms are becoming increasingly popular [5, 14, 19, 23]. Gabapentin is a non-opioid medication, structurally analog of gamma-amino butyric acid, which has been used as an anticonvulsant and antinociceptive drug [9]. In humans, several studies have shown that perioperative gabapentin helps to produce a significant opioid-sparing effect and decreases the postoperative pain score compared with placebo groups [3, 4, 6]. In veterinary medicine, two previous reports showed relevant analgesic effects using gabapentin as an adjuvant for the treatment of hyperalgesia and allodynia in dogs and cats [2, 24]. In contrast, in dogs undergoing amputation of a forelimb, perioperative gabapentin did not provide a significant benefit for postoperative pain management [26]. The aim of this study was to evaluate the analgesic efficacy of gabapentin as an adjuvant for postoperative pain management in dogs. This study was performed following the guidelines of the Brazilian College of Animal Experimentation, and the experimental procedure was approved by an institutional animal care committee (protocol 1182-CEEA). Informed consent was obtained from the owners of all the dogs. Twenty female dogs, aged 6 to 12 years (median, 9.2 years) and weighing between 2.7 and 29.3 kg (median, 10.6 kg), undergoing elective mastectomy of at least 4 mammary glands were enrolled. The preoperative condition of each dog was evaluated through a physical examination and laboratory tests (i.e., complete blood cell count, measurements of the urea, creatinine, alanine aminotransferase and aspartate aminotransferase levels). All the dogs met the following inclusion criteria: American Society of Anesthesiologists physical status of III or less, no arterial hypertension or congestive heart failure, no renal or hepatic dysfunction and no pulmonary metastases. The dogs were randomly assigned to one of the two groups, with 10 animals in each. The dogs were orally (PO) administered with either 10 mg/kg (1 ml/5kg) of gabapentin solution (Neurotin Oral solution, 50 mg/5ml, Pfizer, New York, NY, U.S.A.) (Gabapentin, n=10) or 1 ml/5kg of placebo solution (Placebo, n=10) 120 min prior to surgery. The placebo was produced with the inactive ingredients of the gabapentin oral solution (glycerin, xylitol, purified water and artificial cool strawberry anise flavor.) The postoperative gabapentin or placebo was continued for three days after surgery beginning 12 hr after the preoperative dose. A random number generator (Research Randomizer, Computer software, http://www.randomizer.org/) was used to assign 10 dogs to each of the two groups. After withdrawing food and water for 12 hr and 3 hr, respectively, all dogs were premedicated with 0.03 mg/kg acepromazine (Acepran 0.2%, Univet São Paulo, Brazil) in combination with 0.3 mg/kg morphine (Dimorf, Cristália, Brazil) intramuscularly (IM). Twenty min after the premedication, an intravenous catheter (Insyte, Becton Dickinson, São Paulo, Brazil) was aseptically placed in a cephalic vein, and anesthesia was induced with 4 mg/kg propofol (Propovan, Cristália, Brazil). Orotracheal intubation was performed, and anesthesia was maintained with isoflurane in 100% oxygen using a small animal rebreathing circuit (Samurai, Takaoka, São Paulo, Brazil). Surgical procedures were performed by the same experienced surgeon, using the same surgical technique. The extent of mastectomy was defined as a percentage of mammary tissue excised, an entire mammary gland chain corresponding to a 100% mastectomy [19]. Lactated Ringer's solution (5 to 10 ml/kg/hr) was administered throughout the procedure. All dogs received an initial dose of 0.2 mg/kg meloxicam (Movatec, Roche, São Paulo, Brazil), intravenously, 5 min prior to surgery and additional daily doses of meloxicam (0.1 mg/kg, PO), beginning 24 hr after extubation and continuing for up to 10 days. Based on the anesthetic record, surgery time (time elapsed from the first incision until placement of last suture), extubation time (time elapsed from termination of isoflurane until extubation) and recovery time (time elapsed from the end of anesthesia until standing positon) were recorded for each dog. Extubation was performed when the dog started coughing and/or gagging from the endotracheal tube. The administration of postoperative analgesic drugs was supervised by one anesthesiologist, and the pain assessment was performed by a blinded assessor. The pain level was measured prior to the premedication (baseline scores) and at 0.5, 1, 2, 4, 8, 12, 18, 24, 32, 40, 48, 56, 64 and 72 hr after extubation using the modified Glasgow Composite Measure Pain Scale (GCMPS, 0–10) [15, 23]. In addition, pain was assessed using a Visual Analogue Scale (VAS, 0–100 mm) in which, 0=no pain and 100=worst possible pain, manifested by vocalization, aggression and refusal to allow the examination [18, 19, 23]. Dogs judged to have moderate to severe pain (VAS ≥50 mm or GCMPS ≥3.3) were given morphine (0.5 mg/kg, IM) [23]. At each pain assessment, sedation was scored according to a scale that ranged from 0 to 4, as follows: normal behavior (0); mild sedation, some decrease in normal activity, some resistance to handling, responds to name (1); moderate sedation, easy to handle, still spontaneously active, responds to name, but more slowly (2); moderate to heavy sedation, less spontaneous activity, verbal encouragement needed to rise, minimal resistance to handling (3); sternal recumbency, physical stimulus required to rise, depressed, no resistance to handling (4) [12]. A Kolmogorov–Smirnov test was performed to assess the normality of the variables. Body weight, age, time to endotracheal extubation, duration of surgery and recovery time were compared between groups using the Student's t-test. The Mann-Whitney U test was used to compare additional morphine requirement, pain and sedation scores between groups. The Friedman test was used to compare differences in pain and sedation scores over time within each group. All analyses were performed using GraphPad Instat5. Differences were considered significant when P<0.05. There were no significant differences between groups for demographic data. Mean ± standard deviation of weight, age, time to endotracheal extubation, duration of surgery, recovery time and extent of mastectomy were: 13 ± 10 and 8 ± 8 kg; 8 ± 3 and 10 ± 3 years; 7 ± 5 and 6 ± 2 min; 87 ± 20 and 81 ± 26 min; 225 ± 12 and 213 ± 15 min and 70.5 ± 18 and 68 ± 15% (Gabapentin and Placebo groups, respectively). Histopathologic tumor classification was simple mammary carcinoma (50%), complex mammary carcinoma (37.5%) and amelanotic melanoma (12.5%), with varying degrees of inflammation (slight to moderate). The median pain scores (VAS and GCMPS) did not significantly differ between the treatment groups at any time point. The GCMPS pain scores were higher than the corresponding basal values at 2 hr after extubation in both groups (P<0.0001) (Table 1Table 1.Pain and sedation scores (median and lower-upper range) measured prior to surgery (ie, baseline) and at 0.5, 1, 2, 4, 6, 8, 12, 18, 24, 32, 40, 48, 56, 64 and 72 hr after tracheal extubation in dogs undergoing mastectomy treated with gabapentin (Gabapentin) or placebo (Placebo)TimeVASGMCPSSedation scoresGabapentinPlaceboGabapentinPlaceboGabapentinPlaceboBaseline0 (0–0)0 (0–0)0.08 (0.08–0.08)0.08 (0.08–0.87)0 (0–0)0 (0–0)0.5 hr0 (0–20)0 (0–30)2.43 (0.08–3.81)2.43 (0.08–5.56)3 (0–4)a)3 (1–4)a)1 hr0 (0–10)5 (0–50)2.43 (0.08–3.55)1.99 (0.08–3.81)3 (0–4)a)2.5 (0–4)a)2 hr0 (0–30)20 (0–30)2.43 (0.87–3.55)a)2.53 (0.87–3.55)a)1 (0–3)a)1.5 (0–4)a)4 hr5 (0–30)20 (0–60)1.89 (0.08–3.37)3.08 (0.08–4.55)1 (0–3)a)0 (0–1)8 hr10 (0–50)10 (0–30)1.04 (0.08–5.85)1.35 (0.08–3.66)0 (0–1)0 (0–0)12 hr10 (0–50)5 (0–30)1.43 (0.08–4.05)0.6 (0.08–3.58)0 (0–0)0 (0–0)18 hr5 (0–30)0 (0–30)0.6 (0.08–3.56)0.08 (0.08–3.83)0 (0–0)0 (0–0)24 hr0 (0–30)0 (0–80)0.49 (0.08–3.81)0.08 (0.08–4.99)0 (0–0)0 (0–0)32 hr0 (0–10)0 (0–20)0.08 (0.08–1.46)0.08 (0.08–2.64)0 (0–0)0 (0–0)40 hr0 (0–10)0 (0–20)0.08 (0.08–1.20)0.08 (0.08–1.83)0 (0–0)0 (0–0)48 hr0 (0–10)0 (0–10)0.08 (0.08–1.21)0.08 (0.08–0.08)0 (0–0)0 (0–0)56 hr0 (0–20)0 (0–30)0.08 (0.08–1.46)0.08 (0.08–2.43)0 (0–0)0 (0–0)64 hr0 (0–20)0 (0–10)0.08 (0.08–1.20)0.08 (0.08–2.43)0 (0–0)0 (0–0)72 hr0 (0–20)0 (0–10)0.08 (0.08–1.20)0.08 (0.08–2.33)0 (0–0)0 (0–0)a) Differences from baseline in each group (Friedman test, P<0.05). VAS=Visual Analogue scale (0–100 mm); GMCPS= Glasgow Modified Composite Pain Score (0–10).). Requirement for postoperative rescue analgesia was significantly lower in the Gabapentin than the Placebo group. In the first 24-hr period following surgery, 16 rescue doses of morphine were required for 8 dogs treated with the placebo, whereas only 9 rescue doses were needed for 6 dogs treated with gabapentin (P=0.021) (Table 2Table 2.Number of morphine doses administered to each dog, and total number of morphine doses administered to each treatment group over time in dogs undergoing mastectomy treated with gabapentin (Gabapentin) or placebo (Placebo)GroupsPostoperative time (hr)N° doses0.51248121824324048566472Gabapentin /DogsNo. 1000000000000000No. 2000001000000001No. 3000000000000000No. 4000000010000001No. 5000000000000000No. 6000000000000000No. 7000100100000002No. 8010000100000002No. 9001000010000002No. 10001000000000001Total n° of morphine doses9a)Placebo/DogsNo. 1001000000000001No. 2000010010000002No. 3000000000000000No. 4000000000000000No. 5000011000000002No. 6010011000000002No. 7000010010000002No. 8000010000000002No. 9001000100000002No. 10010010100000003Total n° of morphine doses16a)a) Significant difference between groups (Mann-Whitney U test, P=0.021).). The degree of sedation was similar between the groups. However, compared with baseline values, higher scores were observed until 2 and 4 hr after extubation in the Placebo and Gabapentin groups, respectively (P<0.0001) (Table 1). This study showed that the dogs in the Gabapentin group had a significantly lower incidence of rescue medication than the Placebo group, suggesting a superior level of analgesia when gabapentin was administered perioperatively. VAS and GCMPS pain scores did not differ significantly between groups at any time point. Any real differences between gabapentin and placebo treatments may have been attenuated by the administration of rescue analgesics. The perioperative oral administration of gabapentin reduced the requirement of rescue doses of morphine by 44% in the dogs. These results support the reports of a previous study where perioperative administration of gabapentin resulted in a 50% reduction in the postoperative morphine consumption in women undergoing radical mastectomy [4]. With respect to pain assessment in animals, several scoring systems using a variety of scales (e.g., visual analog, numerical rating, simple descriptive and composite) have been developed and correlated specifically for dogs [7, 15, 18]. In the current study, two methods (i.e., the VAS and GCMPS) were used to obtain a more accurate overall impression of pain [20, 23]. The VAS system is a simple method of scoring pain and is commonly used for both humans and animals. The GCMPS system is internationally recognized and is reported to be as reliable and reproducible as the VAS system for assessing pain in dogs [18]. In addition, in the present study, only one observer scored all the dogs to avoid interobserver variability in the subjective evaluation. Furthermore, a blinded assessor performed the pain assessment. We believed that the postoperative pain was evaluated objectively with minimum variability in the present study. It has been suggested that central neuronal sensitization may play an important role not only in chronic pain states, such as neuropathic pain, but also in postoperative pain [17]. Although the precise mechanism by which gabapentin produces analgesia is unknown, the compound has a high binding affinity for the α2δ subunit of the presynaptic voltage-gated calcium channels, which inhibits calcium influx and subsequent release of excitatory neurotransmitters including glutamate, substance P and noradrenaline [11, 22]. These channels are upregulated in the dorsal root ganglia and spinal cord after surgical trauma [9]. In animal models, gabapentin has been shown to prevent allodynia and hyperalgesia [8, 11]. Other theories regarding the analgesic effects of gabapentin relate to its inhibitory effect on dorsal horn N-methyl-D-aspartic acid receptors [13]. Postoperative pain following mastectomy may induce an inflammatory response that leads to hyperalgesia of the surgical incision with sensitization of peripheral nociceptive nerve terminals and central neurons [14]. For this reason, all dogs were given meloxicam before and postsurgery. When used to control postoperative pain, both nonsteroidal anti-inflammatory drugs (NSAIDs) and gabapentin have been shown to have a significant opioid-sparing efficacy [3, 4, 23]. NSAIDs (i.e., meloxicam) inhibit peripheral and central nervous system prostaglandin synthesis [16], and gabapentin reduces the hyperexcitability of dorsal horn neurons following tissue damage [9]. Clinical trials in humans suggest that the use of the combination of these two non-opioid analgesics promotes superior reduction in opioid requirements [5, 17]. The present study supports these findings and demonstrates that as part of a multimodal analgesic regimen, gabapentin provides a significant effect on analgesic requirements after mastectomy in dogs. Thus, antihyperalgesic drugs, such as gabapentin, may have a role in postoperative pain, and the combination with other antinociceptive drugs may prevent or reduce the peripheral and central sensibilization induced by the surgical procedure, resulting in a decreased need for additional postoperative pain treatment. Unfortunately, there is insufficient information to recommend a specific dose regimen for gabapentin in dogs. In humans, the analgesic efficacy of gabapentin has been associated with 2–3 μg/ml plasma concentrations [25]. Using dogs, Kukanich and Koen [10] studied the pharmacokinetic properties of oral gabapentin at 10–20 mg/kg. The results of this trial showed that plasma concentrations of gabapentin exceeded 2 μg/ml in the majority of dogs only until 8 hr, suggesting that gabapentin was rapidly absorbed and eliminated, and frequent dosing is needed to maintain the minimum targeted plasma concentrations. The dose used in the current study was based on textbook recommendations and recent trials in dogs [1, 26]. Our results showed that 10 mg/kg twice a day of gabapentin did not decrease the pain levels when compared with the placebo treatment, which may be associated with an insufficient dose for postoperative pain control in dogs. Using the same dosing regimen in dogs undergoing hemilaminectomy, Aghighi et al. [1] did not find a detectable reduction in pain scores compared to opioid analgesia alone. This study also reported that in 21.5% and 23.1% of the gabapentin treated dogs, serum concentrations fell below 3μg/ml at 24 hr and 72 hr, respectively, suggesting that the dose and dose interval were too low to obtain stable serum concentrations. The design of this study has some limitations. The variability inherent in this type of a clinical study may have contributed to the lack of significant differences in postoperative pain; it was difficult to control the degree of surgical trauma. The randomization of dogs between treatments resulted in groups that were not completely homogeneous. There was a difference with respect to the type of neoplasia and extent of mastectomy, which may also have affected the intensity of postoperative pain. This variability may have confounded the pain scoring in the present study. Moreover, the number of dogs was also limited, because of the inclusion criteria and need for owner consent. In humans, the major side effects reported from the use of gabapentin include sedation, dizziness and nausea [9]. Although there were no statistical differences in sedation scores between groups, compared with the baseline values, greater sedation scores were observed at 2 hr and 4 hr after extubation in the Placebo and Gabapentin groups, respectively. These findings suggest that gabapentin prolonged the sedation effect, and this result is in agreement with previous studies [3, 6]. Nausea and vomiting were observed in the majority of dogs after the premedication, but this effect is associated with morphine [19]. Thus, it appears that gabapentin did not induce relevant adverse effects. In conclusion, at the dose and frequency used in this study, perioperative gabapentin reduced the requirement of postoperative rescue analgesia in dogs after mastectomy.

TITLE: Effects of low-dose ketamine on succinylcholine-induced postoperative myalgia in outpatient surgeries: a randomized, double-blind study ABSTRACT.OBJECTIVE: Despite the many complications of succinylcholine, it is still widely used as a superior muscle relaxant for rapid sequence induction. One of these complications is postoperative myalgia (POM). The aim of this study was to investigate the prophylactic effect of low-dose ketamine on the incidence and severity of POM. ABSTRACT.MATERIALS AND METHODS: In this double-blind clinical study, a total of 148 patients scheduled for general anesthesia were randomly divided into two equal groups. Initially, in Group K, 0.5 mg/kg of ketamine was injected intravenously, whereas in Group N, the same volume (5 mL) of normal saline was injected. Thereafter, anesthesia was induced in all patients, by injecting 1.5 mg/kg of fentanyl and 2 mg/kg of propofol intravenously. Following the loss of eyelid reflex, 1.5 mg/kg of succinylcholine was injected intravenously as a muscle relaxant and then the patients were intubated. POM was defined as a pain with no surgical interferences, and its intensity was graded based on a four-point scale. The incidence and severity of myalgia were assessed by a blinded observer 24 hours after surgery. ABSTRACT.RESULTS: In terms of demographic data, the results of this study showed that there is no significant difference between patients in both groups (P>0.05). Overall, the incidence of POM in Group K was significantly less, when compared with Group N (P<0.05), but both groups were comparable based on the grade 2 of POM. After the induction of anesthesia, the systolic and diastolic blood pressure values were found to reduce in both groups (P<0.05). However, the changes were somehow similar, and repeated measures of variance analysis showed no significant difference in the two study groups (P>0.05). ABSTRACT.CONCLUSION: The addition of 0.5 mg/kg of ketamine to propofol for the induction of anesthesia can be effective in reducing the incidence of low-grade POM. BODY.INTRODUCTION: For >60 years, succinylcholine is still being administered as a selective relaxant for rapid sequence intubation by anesthesiologists in many countries.1 It has been shown to possess unique features such as low cost, fast-acting, short half-life, safe metabolites, and causing excellent muscle relaxation for intubation;2 however, it has many side effects as well. Postoperative myalgia (POM), with an incidence rate of ~41%–92%, is one of the most common side effects of this drug and can take several days to cause significant discomfort in patients.3 However, its effect is felt more in the throat, neck, shoulder, and abdominal muscles and is common among patients with outpatient surgery.4 Due to its unknown real context of pathogenesis and in an effort to reduce the incidence and severity of succinylcholine-induced myalgia, various medications including nondepolarizing muscle relaxants, benzodiazepines, magnesium sulfate, opioids, gabapentin, and nonsteroidal anti-inflammatory drugs have been tested, with varying degrees of success.5,6 Ketamine is an N-methyl-d-aspartate (NMDA) receptor antagonist with excellent analgesic activity, and at subanesthetic doses it is capable of preventing central sensitization, hyperalgesia, drug resistance creation, and reduction of postoperative pain.7 Although, ketamine administration could produce unpleasant psychogenic complications, no disagreeable effects have been recorded in patients who received a low dose of ketamine. The effects of ketamine on postelectroconvulsive therapy (ECT) myalgia have been studied earlier, and the results showed that ketamine could not prevent this type of myalgia.8 To the best of our knowledge, the effect of ketamine on the prevention of POM is yet to be determined. Therefore, in this randomized, double-blind study, the prophylactic effect of low-dose ketamine on the incidence and severity of myalgia caused by succinylcholine injection in patients undergoing outpatient surgery was investigated. BODY.MATERIALS AND METHODS: This study was approved by the Ethics Committee of Kurdistan University of Medical Sciences and was registered in the Iranian Registry of Clinical Trials (IRCT2014062412789N5). A complete description of the study was also presented to each participant, and written informed consent was obtained. The sample size was calculated based on the assumption that the incidence of POM in outpatient cases is ~70%, and intervention that can cause 25% reduction in incidence of POM will be interesting. To consider this difference and type I error equal to 5% and 90% power of the study, 72 patients were required to be in each group (α=0.05 and β=90%), but in order to avoid possible loss of samples during the study, the number of patients in each group was increased to 74. Thus, 148 patients, who were scheduled for outpatient surgery under general anesthesia, belonging to the American Society of Anesthesiologists physical status I and II within the age of 18–70 years, were included in this double-blind randomized clinical trial. Patients with a history of allergy to medications; substance abuse; malignant hyperthermia; myopathy; cardiovascular, liver, and advanced kidney diseases; and the risk of difficult intubation based on physical examination were excluded from the study. Before surgery, patients were evaluated by the anesthesiologists. The study protocol and evaluation of myalgia based on Kararmaz's criteria were described to the patients.4 Based on a computer-generated random sequence, the 148 patients were enrolled into one of the two groups (ketamine or saline). The patients did not receive premedication. In the operating room, standard monitoring of the noninvasive blood pressure, electrocardiogram, heart rate, and pulse oximetry was done, and the preliminary values were recorded. Thereafter, a venous cannula (G=18) was placed on the dorsum of a patient's hand. Before the induction of anesthesia in patients of Group K, 0.5 mg/kg of ketamine (a 5 mL volume was reached by adding distilled water) was injected intravenously and 5 mL of normal saline was injected intravenously slowly into patients in Group N. Drugs were prepared in 5 mL syringes by an anesthesia nurse who was unaware of the grouping. After injecting the study drugs, 1.5 mg/kg of fentanyl was injected intravenously within 60 seconds and subsequently 2 mg/kg of propofol was administered intravenously within 30 seconds for the induction of anesthesia. Following the loss of eyelid reflex, 1.5 mg/kg of succinylcholine was injected intravenously and patients were ventilated using bag and mask and with 100% oxygen. After fasciculation, the values of heart rate and blood pressure were measured and recorded, and tracheal intubation was performed. The maintenance of anesthesia continued using a mixture of oxygen, N2O (40/60), and isoflurane 1 MAC. After 5 minutes of tracheal intubation, the values of heart rate and blood pressure were obtained and recorded again. For maintenance of muscle relaxation, 0.2 mg/kg of atracurium was used as suggested by the surgeon. At the end of the surgery, muscle relaxation was reversed using neostigmine and atropine. After the desired spontaneous ventilation, the patients were extubated. The patients were transferred to the recovery room and complications, if any, were recorded during recovery. After meeting the discharge criteria, the patients were discharged to be taken home and cared for by a responsible adult. The incidence and severity of myalgia in the patients were determined 24 hours after surgery by a medical student who was unaware of the grouping. Myalgia is defined as "a pain with no surgical interference" and is graded based on Kararmaz et al's4 four-point scale as follows: 0= no muscle pain, 1= muscle stiffness limited to one area of the body, 2= muscle pain or stiffness noticed spontaneously by a patient who requires analgesics, and 3= incapacitating generalized, severe muscle stiffness or pain. Statistical analysis was conducted using SPSS Version 12.0 software (IBM Corporation, Armonk, NY). The comparison profile of the patients was performed using the analysis of variance test. The incidence and severity of myalgia were compared using the chi-square and independent t-tests. The value of P<0.05 was considered statistically significant. BODY.RESULTS: In this study, a total of 207 patients were scheduled for outpatient surgery from July 2013 to August 2014. Of them, 29 patients could not meet the entry criteria. Twenty-seven patients had no willingness to participate in the study, and the surgery of three patients was canceled. The remaining 148 patients were randomly assigned into two groups. Two patients in Group N due to nausea, vomiting, and pain and one patient in Group K due to arrhythmia were turned from outpatient to inpatient. Two persons from Group K did not cooperate with the evaluation of POM. The data associated with the 72 patients in Group N and 71 patients in Group K were analyzed (Figure 1). There were no significant differences in terms of age, sex, and duration of surgery between both groups (Table 1). In Group K, 13 (18.1%) out of the 71 patients had myalgia, whereas 36 (50%) out of the 72 patients had myalgia in Group N (P=0.001). Grade 1 POM was lower in Group K when compared with Group N (nine in Group K versus 33 in Group N; P<0.001), whereas the incidence of grade 2 POM was comparable among patients of the two groups (Table 2). The baseline values of systolic and diastolic blood pressure and heart rate in both groups were similar. After the induction of anesthesia, the values of systolic and diastolic blood pressure decreased in both groups (P<0.05). However, these changes were somehow similar in the two groups, and repeated measures of variance analysis showed no significant difference in both groups (P>0.05). Changes in heart rate after induction and intubation between the two groups were compared and repeated measures of variance analysis showed no significant difference in the two groups (P>0.05; Table 3). BODY.DISCUSSION: The results of this study showed that ketamine significantly reduced the incidence of succinylcholine-induced myalgia. Succinylcholine is a muscle relaxant that is commonly used due to deep neuromuscular blocks for intubation in patients undergoing ambulatory anesthesia, but associated myalgia has been shown to occur in 41%–92% of patients.9,10 Since ambulation increases the possibility of myalgia and its intensity, ambulatory patients are suitable for investigating this complication. Therefore, this group of patients was chosen for the study. Myalgia is most prevalent following the use of succinylcholine in the first day of surgery.11 As shown in a study, 92% of patients reported myalgia in the first 24 hours of study and the incidence of myalgia did not differ at 24 hours and 48 hours after surgery.8 In this study, myalgia was evaluated only in the first 24 hours after surgery. It seems that the intrafusal contraction of muscle fibers caused damage to spindles and subsequently myalgia.12 In vitro studies have shown that active, excessive, and continuous muscle contractions increase the uptake of calcium, activation of phospholipase A2, arachidonic acid, and prostaglandins production, thereby increasing the risk of muscle damage and pain.13 The results of this study showed that 50% of the patients in Group N experienced myalgia after anesthesia was induced with propofol. In a meta-analysis, the average incidence of myalgia in the first 24 hours with thiopental was 49.2% and with propofol was 65.4%.14 The incidence of myalgia in the present study was slightly lower when compared with the aforementioned meta-analysis. It appears that participants in this study belonged to low-level POM in general. Ketamine creates a complex combination of both antinociceptive and pronociceptive actions. In fact, based on the mechanisms of analgesia with a direct receptor, the analgesic effect of ketamine is associated with the plasma levels of drugs and pain reduces with subanesthetic doses of ketamine.15,16 In general, it seems that the NMDA receptor plays an important role in the pathophysiology of pain, and the Supra spinal block of the NR2B NMDA subunit by ketamine has an important antinociceptive effect.17 Recent studies have shown the role of NMDA receptors in facilitating the process of pain in the central nervous system, this receptor is also responsible for central sensitization and windup phenomenon. The administration of NMDA receptor antagonists prevents the development of sensitization, hyperalgesia, and drug resistance.18 Ketamine is an antagonist of this receptor and has excellent analgesic activity at doses under anesthesia.19 It strengthens the performance of mu and kappa opioid receptors and also has direct effects on the delta opioid receptor. As such, ketamine certainly modulates the response of opioid receptors and reduces tolerance to opioids.20 It also strengthens the endogenesis of antinociceptive systems, in part, by its aminergic (serotonergic and noradrenergic) activation and inhibition of reuptake.21 It inhibits the synthesis of nitric oxide, which probably contributed to the analgesic effect.22 Also, there is evidence suggesting that ketamine interferes with nicotinic, muscarinic, and monoaminergic receptors. As a result of the undesirable and unwanted side effects, ketamine usage is controversial in nonanesthetized patients, but when surgery is done under general anesthesia, these side effects are barely seen.19 In this study, surgery was performed under general anesthesia and none of the patients experienced delirium and nightmare. In our previous study,8 the effect of prophylactic ketamine on myalgia after ECT in 50 patients with major depression was demonstrated, in which 1 mg/kg of propofol and 0.3 mg/kg of ketamine were used for the induction of anesthesia, and muscle relaxation was induced using 0.5 mg/kg of succinylcholine. The results showed that the addition of ketamine to propofol had no effect on the incidence of myalgia after ECT, and only 12% of the patients had myalgia within 24 hours after ECT. However, our previous study8 is different in some aspects from the present study. In our previous study, ECT patients were investigated and the cause of myalgia was found to be different in surgical patients. Second, unlike the surgery, myalgia intensity in ECT is not related to fasciculation and motor activities.23 Third, the dose of anesthetic drugs for the induction of anesthesia and muscle relaxation was less than that of the present study. The result of our previous study, which was carried out on patients undergoing ECT, is not in line with the present study. Cardiovascular changes were similar after the induction of anesthesia in both groups. In theory, hemodynamic stability can be predicted based on the induction of anesthesia with propofol and ketamine, when compared with propofol alone. This is because ketamine increases blood pressure and heart rate; therefore, the addition of this drug to propofol during anesthesia induction can inhibit the reduction of these parameters before the stimulation of laryngoscopy and tracheal intubation. Also, following laryngoscopy and tracheal intubation, the synergistic effect of these two drugs can increase the depth of anesthesia and result in smoother cardiovascular response to airway stimulation. It appears that 0.5 mg/kg of ketamine could not produce such an impact on the hemodynamic system, thereby resulting in a significant effect. BODY.STUDY LIMITATIONS: One of the limitations of this study is the insufficient time for myalgia evaluation (24 hours). Failure to assess the incidence and severity of fasciculation is another limitation of this study. In this study, ketamine was diluted with sterile water, although this is not a wrong practice, but further addition of water, as carried out in this study, could produce ketamine with concentration below the physiological osmotic pressure, which may interfere with the effects of ketamine. BODY.CONCLUSION: The addition of 0.5 mg/kg of ketamine to propofol for the induction of anesthesia can be effective in reducing the incidence of POM, without any change in hemodynamic indices.

TITLE: Applicability Evaluation of Simplified Cognitive Behavioral Therapy ABSTRACT.BACKGROUND: We have developed a structured cognitive behavioral therapy manual for anxiety disorder in China, and the present study evaluated the applicability of simplified cognitive behavioral therapy based on our previous research. ABSTRACT.AIMS: To evaluate the applicability of simplified cognitive behavioral therapy (SCBT) on generalized anxiety disorder (GAD) by conducting a multi-center controlled clinical trial. ABSTRACT.METHODS: A multi-center controlled clinical trial of SCBT was conducted on patients with GAD, including institutions specializing in mental health and psychiatry units in general hospitals. The participants were divided into 3 groups: SCBT group, SCBT with medication group and medication group. The drop-out rates of these three groups, the therapy satisfaction of patients who received SCBT and the evaluation of SCBT from therapists were compared. ABSTRACT.RESULTS: (1) There was no significant difference among the drop-out rates in the three groups. (2) Only the duration and times of therapy were significantly different between the two groups of patients who received the SCBT, and the therapy satisfaction of the SCBT group was higher than that of the SCBT with medication group. (3) Eighteen therapists who conducted the SCBT indicated that the manual was easy to comprehend and operate, and this therapy could achieve the therapy goals. ABSTRACT.CONCLUSION: The applicability of SCBT for patients with GAD is relatively high, and it is hopeful that SCBT can become a psychological treatment which can be applied in medical institutions of various levels. BODY.1. INTRODUCTION: The core features of generalized anxiety disorder (GAD) are the chronic and persistent loss of self-control due to worries, and a cognitive evaluation bias towards threats and risks, which causes significant impairments in work, interpersonal social life, physical health, mental health and so forth.[1,2] Currently, the main treatment for GAD is medication with psychotherapy as the supplement.[3,4] The most commonly used form of psychotherapy to treat anxiety is cognitive behavioral therapy (CBT), and it has been shown to be more effective for the treatment of mild anxiety than other psychotherapies.[5-7] Brief cognitive behavioral therapy (BCBT) is a short-term psychotherapy which has gained more attention internationally in recent years, and clinical trials have shown it's effectiveness in treating anxiety disorder.[8,9] In the studies done outside of China, the manual for BCBT was gradually developed and applied in primary medical institutions. However, there are few systematic, detailed and structured manuals provided to medical workers for use in China. Previously, we developed a structured simplified cognitive behavioral therapy (SCBT) manual that could be used by medical workers providing services at district level institutions or those who had not received systematic training. We reported on the effectiveness of this treatment which, at the time, was quite early in the provision of psychological services in China.[10-12] The present study was based on this previous research[10,11] to provide further evaluation of the SCBT technique. BODY.2. METHODS.2.1 SCBT MANUAL: There are four sections in the manual: (a) instructions, (b) procedure and content, (c) specific treatment content and (d) addendum.[10] Four key steps are emphasized in every treatment session: feedback on the previous homework, treatment content, relaxation technique exercises and homework assignments. BODY.2. METHODS.2.2 PARTICIPANTS.2.2.1 SAMPLE: Participants were recruited through posters, nurse registrations and outpatient doctors' recommendations. The time frame of the recruitment was from March, 2014 to December, 2015. Participants were recruited from the following locations: Shanghai Psychological Counselling and Treatment Center, Tenth People's Hospital of Tongji University, Shanghai Hongkou Mental Health Center, Shanghai Yangpu Mental Health Center and Nanjing Brain Hospital affiliated to Nanjing Medical University. BODY.2. METHODS.2.2 PARTICIPANTS.2.2.2 INCLUSION CRITERIA: 1 Participants who met the diagnostic criteria for GAD according to the DSM-IV; 2 participants whose HAMA-14 scores were equal to 14 or above, and equal to 29 or below; and participants whose GAD-7 scores were equal 5 to or above; and participants whose HAMD-17 scores were below 14; 3 females and males between 18 and 65; 4 participants whose education levels were equal to elementary school or above; 5 participants whose visual and audio levels were high enough to complete the assessments required; 6 participants who had not received psychological counselling or treatments in any form in the past three months; 7 participants who were willing to participate and provided written informed consent. BODY.2. METHODS.2.2 PARTICIPANTS.2.2.3 EXCLUSION CRITERIA: 1 Participants who had a current severe medical condition; 2 participants who had severe suicidal ideation; 3 females who were pregnant or lactating; 4 participants who had comorbid psychosis or had psychotic symptoms, psychoactive substance dependence or abuse, personality disorder, or mental retardation; 5 participants whose symptoms did not improve after receiving medication with sufficient doses and durations and psychotherapies. BODY.2. METHODS.2.3 RESEARCH TOOLS: 1 Adapted patient satisfaction questionnaire [8] evaluating the acceptability of SCBT. There are 8 items using a scale of 1 to 5. 2 Adapted manual evaluation scale [8] evaluating the applicability of SCBT. There are 3 sub-scales, as stated below: (a) the treatment feedback scale for therapists after each treatment: therapists evaluate the content, goals, flexibility, effectiveness and other aspects of each treatment. There are 8 items using a scale of 1 to 7. (b) the treatment feedback scale for therapists after each week: therapists evaluate the effectiveness, applicability and other aspects of treatments after each week. There are 8 items using a scale of 1 to 7. (c) the feedback summary scale for therapists: the main focus is for therapists to evaluate SCBT in terms of their general practice and experience. There are 8 items using a scale of 1 to 7. BODY.2. METHODS.2.4 STUDY DESIGN: Randomized parallel control and blind evaluation methods were employed in the present study. The present study used a random table generated by computer, and participants were given random numbers according to the order of their enrollment. However, some participants did not cooperate with the group assignment or switched to another group during the study due to worries about the side-effects of drugs, thinking that psychotherapy was useless and being influenced by the information of group assignments, therefore the study was only partially randomized; in fact, the whole randomized design was affected. Participants were divided into three groups: the SCBT group, the SCBT with medication group and the regular medication group. Based on the previous related literature, we planned to recruit 30 participants into each group. Within the sample of 112 participants included in the analysis, 32 (28.6%) of them did not cooperate with the group assignment or switched to another group. Blind evaluation method means that the people who conducted the assessments did not know which group participants were assigned to. BODY.2. METHODS.2.5 STUDY PROCEDURE.2.5.1 SIMPLIFIED COGNITIVE BEHAVIORAL THERAPY GROUP (SCBT GROUP): Participants in this group only received SCBT during the treatment period, and did not receive any other treatment in any form. The treatment duration was 8 weeks with 12 structured sessions. In the first 4 weeks: twice a week; in the latter 4 weeks: once a week. Every individual therapy session lasted for an hour, and the evaluations were conducted at the baseline and the end of the eighth week. BODY.2. METHODS.2.5 STUDY PROCEDURE.2.5.2 COMBINED TREATMENT GROUP (SCBT WITH MEDICATION GROUP): Participants in this group received SCBT with medication during the treatment period. The categories of medications were mainly required to be SSRI, SNRI or benzodiazepines. The evaluation schedule was the same as the SCBT group's. BODY.2. METHODS.2.5 STUDY PROCEDURE.2.5.3 REGULAR MEDICATION GROUP (MEDICATION GROUP): Participants in this group received routine medication only, and did not receive any SCBT. The categories of medications and evaluation schedule were the same as those of the SCBT with medication group. BODY.2. METHODS.2.5 STUDY PROCEDURE.2.5.4 THE APPLICABILITY EVALUATION OF SCBT: The applicability of SCBT mainly includes acceptability and feasibility.[8] Acceptability is mainly referred to as patients benefitting from the treatment, whether or not they would recommend it to others, their satisfaction towards the treatment and so forth. The adapted patient satisfaction questionnaire was used to evaluate this factor. Feasibility mainly includes the drop-out rate, and the compliance and feedback of therapists to the manual; the adapted manual evaluation scale was employed to assess this factor.[8] The Likert Scale's rating system was employed to rate the patient satisfaction questionnaire and manual evaluation scale.[13] BODY.2. METHODS.2.6 STATISTICAL METHODS: As the acceptability and partial feasibility were evaluated for the whole intervention, only the data of participants who completed the intervention were analyzed. There were participants who dropped out of the study, and their data were excluded from the analysis. Furthermore, missing data were not processed or included in the analysis. Therefore, the present study employed Per Protocol Set Analysis, which meant only analyzing data of participants who completed the intervention. SPSS 17.0 was used for statistical analysis. Enumeration data were described with frequencies and rates. Independent sample t tests, multiple tests and X2 tests were employed. The value of p being smaller than 0.05 indicated that the difference was statistically significant. BODY.3. RESULTS: With Per Protocol Set Analysis being used, there was a total of 112 participants who were included in the final analysis (SCBT group: 34, SCBT with medication group: 45, medication group: 33). The three groups were not significantly different in age, gender, education level or marital status (F=1.88, df=2, p=0.158; X2=1.16, df=2, p=0.560; X2=3.50, df=6, p=0.744; X2=5.08, df=6, p=0.454). BODY.3. RESULTS.3.1 ACCEPTABILITY EVALUATION OF SCBT: This part of the analysis only accepted data of participants who received SCBT and completed the eight-week-long intervention (i.e., 34 participants from the SCBT group and 45 participants from the SCBT with medication group). As stated in Table 1, participants in the SCBT group and SCBT with medication group were not significantly different in satisfaction factors, such as the quality of treatment, the duration and the number of session, or whether their expectations for treatment had been met. BODY.3. RESULTS.3.2 FEASIBILITY EVALUATION OF SCBT.3.2.1 DROP-OUT RATE: As stated in Table 2, there was no significant difference among the drop-out rates of the three groups (X2=1.41, df=2, p=0.494) with the drop-out rate of the medication only group being the highest. The summation of drop-out rates of the SCBT group and SCBT with medication group was 24.8%. BODY.3. RESULTS.3.2 FEASIBILITY EVALUATION OF SCBT.3.2.2 EVALUATIONS OF THE SCBT MANUAL BY 18 THERAPISTS: Therapist evaluations of each treatment are shown in Table 3. Based on the content and experience of each treatment, therapists conducted evaluations on each item of each participant. All therapists indicated that the flexibility allowed by the manual was medium in each treatment, and the flexibility of the second treatment was the lowest as there was basically no flexibility. Table 4 shows the therapists' evaluation of each week's treatment. Similarly, therapists evaluated the item for each participant based on the content and experience of each weeks treatment. All therapists indicated that most content of the weekly treatment was useful, that each week's assignment could be used effectively and that the feasibility was good. Overall evaluation of the program by the therapists is shown in Table 5. Through feedback summary scales of therapists, every participant who completed the eight-week-long intervention was evaluated on every item based on each therapist's insights into the 12 interventions. Therapists suggested that this manual was basically suitable to use on patients. BODY.4. DISCUSSION.4.1 MAIN FINDINGS: SCBT follows the principle of starting simple and gradually becoming more advanced. The main focus of treatment is for patients to identify, analyze and modify maladaptive thoughts and behaviors. In this regard the core technique of CBT (i.e., cognition restructuring and exposing) is included in SCBT, which is consistent with conventional CBT and BCBT.[9,14] The manual for SCBT was edited in a structured way. The duration of treatment is relatively shorter, and the number of sessions is fewer, making it easier for novice therapists to master and conduct. Therefore SCBT can be applied in primary medical centers and is consistent with the lower requirements of therapists conducting BCBT.[8,10] The drop-out rates for the three groups were not significantly different, but drop out from the medication only group was the highest. This was mainly because that in contrast to the medication only group, participants of in the SCBT group and SCBT with medication group developed good working communities and stable treatment relationships by receiving 12 treatments. The drop-out rate of all participants who received SCBT (SCBT group and SCBT with medication group) was 24.8%, which is comparable to conventional CBT's and BCBT's rates.[8,15] Therapists considered treatments to be somewhat flexible, and this was mainly because the manual was structured and therapists had little experience. Among the 12 sessions, the flexibility of the second session was rated as the lowest. This was due to the fact that the second treatment session used narrative therapy. In that particular session participants wrote down content and therapists reflected main factors of these depictions back to them. The evaluation of sessions by the therapists reflected that treatment was rushed to achieve each week's goal. This was also found to be true in other studies of BCBT.[8,16] The reason for this is that therapists are accustomed to conducting conventional CBT and they may feel that 12 simplified treatments are not enough. This suggests that further simplifications and adjustments to SCBT may be needed, and every treatment needs to be organized more reasonably, which can enable participants to master the core technique of CBT within 12 treatments.[17] BODY.4. DISCUSSION.4.2 LIMITATIONS: Participants were not assigned randomly, so it is difficult to generalize the conclusion that the applicability of SCBT is high. In addition, not analyzing the data of participants who dropped out may exaggerate the feasibility and acceptability of SCBT. BODY.4. DISCUSSION.4.3 IMPLICATIONS: On the basis of previous studies, the present study tested the applicability of SCBT, and provided methodological evidence on treating anxiety disorder for Chinese primary medical workers.

TITLE: Co-morbidities, complications and causes of death among people with femoral neck fracture – a three-year follow-up study ABSTRACT.BACKGROUND: The poor outcome after a hip fracture is not fully understood. The aim of the study was to describe the prevalence of co-morbidities, complications and causes of death and to investigate factors that are able to predict mortality in old people with femoral neck fracture. ABSTRACT.METHODS: Data was obtained from a randomized, controlled trial with a 3-year follow-up at Umeå University Hospital, Sweden, which included 199 consecutive patients with femoral neck fracture, aged ≥70 years. The participants were assessed during hospitalization and in their homes 4, 12 and 36 months after surgery. Medical records and death certificates were analysed. ABSTRACT.RESULTS: Multivariate analysis revealed that cancer, dependence in P-ADL (Personal Activities of Daily Living), cardiovascular disease, dementia at baseline or pulmonary emboli or cardiac failure during hospitalization were all independent predictors of 3-year mortality. Seventy-nine out of 199 participants (40 %) died within 3 years. Cardiovascular events (24 %), dementia (23 %), hip-fracture (19 %) and cancer (13 %) were the most common primary causes of death. In total, 136 participants suffered at least one urinary tract infection; 114 suffered 542 falls and 37 sustained 56 new fractures, including 13 hip fractures, during follow-up. ABSTRACT.CONCLUSION: Old people with femoral neck fracture have multiple co-morbidities and suffer numerous complications. Thus randomized intervention studies should focus on prevention of complications that might be avoidable such as infections, heart diseases, falls and fractures. BODY.BACKGROUND: Hip fractures are a common and major health problem among older people [1]. The annual number of such fractures in Sweden is expected to almost double during the first half of this century [2]. There is a well-established increased risk of death after hip fracture [3, 4]. It has been shown that older people have a 5- to 8-fold increased risk of dying during the first 3 months after a hip fracture [4]. Studies have been performed to optimize care of hip fracture patients and the consensus concerning preoperative management, time to surgery, operative management, surgical technique and postoperative care has led to recommendations concerning clinical care pathways and a multidisciplinary approach [5–8]. Despite research into care improvements for hip fracture patients, for example in the fields of medical and surgical care or the use of multidisciplinary teams, it has been shown that fewer than half regain their previous level of function [9] and the mortality rate has remained stable over the past 40 years [10]. A variety of postoperative complications and times to follow-up are described. One study found that 33 % of the participants had at least one complication after an operation for hip-fracture which led to prolonged hospitalization. The most common postoperative complications were delirium and infection [11]. Another study found heart failure and chest infections to be the most common postoperative complications [12], while a more recent study showed that falls, fractures and pneumonia were the most common [13]. A range of interventions to reduce the rate of in-hospital postoperative complications and mortality have been reported in the literature. One such study found that postoperative complications and 12-month mortality among community dwellers were reduced when a comprehensive multidisciplinary fast-track treatment and care program were put in place [14]. We have shown earlier that a multidisciplinary, multi-factorial rehabilitation program reduced in-hospital complications [15], including significant fewer in hospital falls but there was no difference in the number of falls during 1 year after discharge [16, 17]. Death following hip fracture has been associated with several risk factors; older age and male sex, severe systemic disease, pre-fracture functional impairment, cognitive decline, coronary heart disease and the number of co-morbidities [18–23]. The causes of excess death have been a subject of debate. One study suggests that the increased mortality is associated with postoperative complications [24], others ascribe it to pre-fracture co-morbidities together with postoperative complications [12, 19], or suggest that the co-morbidities are the underlying cause [25]. Only a few studies describe the causes of death [24, 26–29]. The most common causes of death in one autopsy study were chest infection, cardiac failure, myocardial infarction and pulmonary embolism [29]. Among nursing-home residents with hip fracture the most common causes of death were infection, dementia and cardiac events [26] and, in more recent studies, cardiac and infectious diseases [27, 28]. In spite of earlier research, the poor outcome for people with hip fracture has not improved and mortality has not been reduced. Since neither the events leading to death, nor the causes of death or the patient's outcome after discharge have been fully investigated, we decided to explore these factors in order to discover factors that might be adjusted in order to improve outcome. Thus, the aim of this study was to describe the prevalence of co-morbidities, complications and causes of death and to investigate factors that could predict mortality in old people with femoral neck fracture. BODY.METHODS.STUDY DESIGN: The article is based on data from a randomized controlled trial, evaluating a multidisciplinary intervention program for persons with a femoral neck fracture compared to conventional care. The intervention resulted in fewer complications during hospitalization, but there were no differences in the incidence of complications and mortality between the intervention and control groups after discharge (data not shown). The participants are analysed as one group in the present study, they were followed from admission to hospital until death, relocation or the end of 36 months of follow-up. The description of the recruitment and randomization as well as the content of the intervention has been presented in detail in earlier articles [15, 16]. All participants received oral and written information and in those cases where they were not able to answer themselves their next of kin was also asked. A written informed consent for participation in the study was required. BODY.METHODS.SAMPLE: The study included 199 participants with femoral neck fractures (index hip fracture) aged 70 years or older, consecutively admitted to the Orthopaedic Department at the University Hospital in Umeå, Sweden, over 32 months. Flow chart of the results of all 353 patients with femoral-neck fractures during the study period is shown in Fig. 1.Fig. 1Flow chart of the results of all 353 patients with femoral-neck fractures during the study period Exclusion criteria were: the presence of rheumatoid arthritis, severe hip osteoarthritis, and pathological fracture, due to the planned operation methods to be used in the study. Patients with severe renal failure, those who were bedridden prior to the fracture and patients already admitted to hospital for other reasons, who then fell and suffered a hip fracture were also excluded. The surgeon decided whether or not an operation planned according to the study protocol was appropriate for the patient. BODY.METHODS.DATA COLLECTION: Trained research nurses, not involved in the patients' medical care or nursing, assessed the patients during hospitalization and at 4, 12 and 36 months subsequently. At the follow-ups the nurse was accompanied by a physiotherapist or an occupational therapist who also made assessments. The patients' general health was assessed before surgery, according to the American Society of Anaesthesiologists (ASA) classification [30], by the attending anaesthesiologist. ASA 1 indicates a healthy person; ASA 2, a person with a mild systemic disease; ASA 3, a person with severe systemic disease; ASA 4, a person with an incapacitating disease that is a constant threat to life; ASA 5, a moribund person who is not expected to live 24 h with or without surgery. In the analysis the ASA results were categorized as ASA 1–2 and ASA 3–4. No person classified as ASA 5 was included. All medical and social data were collected from interviews with participants, relatives and staff, and included morbidity and complications. Data were also collected from the medical and nursing records. A broad definition of complications was chosen to describe the wide spectrum of complications. The complications were classified dichotomously (present/absent) and the total number of times a complication occurred was counted. The participants with cardiac failure at baseline were judged to have cardiac failure as a complication during follow-up if they had been treated by a medical doctor due to exacerbation of the disease. Cardiac failure and myocardial infarction are referred to in the text as cardiovascular events. Infections were divided into 5 groups; chest infections, urinary tract infections, superficial and deep wound infections and other infections. A fall was defined as an incident when the participant unintentionally came to rest on the floor or ground and also included syncopal falls [31]. For those participants who died during the study, medical records were reviewed and all complications and the "history of death" were noted. If a participant died between follow-ups, complications that occurred between last follow-up and death were forwarded, which means they were registered at the next follow-up. The primary and secondary causes of death were also obtained from the death certificates. In Sweden every citizen has a unique 10-digit personal identification number which makes it possible to obtain cause and date of death from the Centre for Epidemiology (EpC), National Board of Health and Welfare, Sweden. The diagnoses are coded according to the International Classification of Diseases (ICD), which is validated and used as an international standard by the WHO (World Health Organization). In Sweden 99 % of deceased receive a diagnosis [32]. If a person died within 30 days after a fracture the primary cause of death was coded, according to international standards, as related to the trauma irrespective of the actual cause of death. For example, a myocardial infarction complicating a hip fracture would be coded as a secondary cause of death. In our analyses the causes of death were determined through assessment of the notes concerning the person's death and the information obtained from death certificates. Fifteen of the causes of death stated in the death certificates were changed after examination of the patients' medical records. Two of these fifteen were changed in accordance with autopsy reports, another three after more information was retrieved and a further ten were changed to secondary cause on the death certificate. For example, a participant with advanced dementia who suffered terminal pneumonia was judged to have died due to dementia rather than pneumonia. A geriatrician, unaware of study-group allocation, analysed all assessments and documentation once the study was ended, to determine whether the participants fulfilled the DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition) criteria [33] for dementia, delirium and depression. The Ethics Committee of the Faculty of Medicine at Umea University approved the study (§ 00–137). BODY.METHODS.STATISTICS: Pearson's chi-square test, Fisher's exact test and Mann–Whitney U test were used to analyse differences between the deceased and the survivors regarding complications. Univariate and multivariate Cox proportional hazard regression was used to analyse associations between baseline variables and complications during hospitalization and all-cause mortality during the 3-year follow-up. All baseline variables and complications during hospitalization associated with time to death (p < 0.05) in univariate analyses were included in two separate multivariate models (Model A and B in Table 1). Correlations between baseline variables and among complications during hospitalization were tested using Pearson's and Spearman's coefficients; the covariate Living in residential care facilities before fracture was removed due to its strong correlation with Dependence in personal activities of daily living (P-ADL) (r > 0.6). Stepwise backward deletion was performed manually until only significant variables remained in the models.Table 1Baseline characteristics and complications among participants and differences among deceased and survivors. Univariate and multivariate cox regression analysesAll casesDeceased within 3 yearsSurvivorsUnivariateMultivariate n = 199 (%) n = 79 n = 120Pa Pb HRCIBaseline characteristics:Model A.Age, mean ± SD82.2 ± 6.282.9 ± 5.881.7 ± 6.50.2330.4351.0160.977–1.056Female148 (74 %)53950.0290.0520.6040.363–1.005Living in residential care facilities73 (37 %)37360.028Living alone146 (74 %)59870.696Current smoker (n = 169)6 (4 %)240.955Dependence in walking (n = 195)25 (13 %)15100.004Dependence in P-ADL115 (58 %)5956<0.0010.0052.3791.300–4.354Cancer (n = 194)29 (15 %)218<0.001<0.0013.4492.020–5.890Cardiovascular disease (n = 194)110 (57 %)5555<0.0010.0042.1771.287–3.682Dementia64 (32 %)37270.0010.0161.9211.131–3.262Depression (n = 197)78 (40 %)39390.040Diabetes (n = 197)40 (20 %)22180.059Kidney disease (n = 194)21 (11 %)10110.321Pulmonary disease (n = 194)33 (17 %)17160.049Stroke (n = 195)49 (25 %)23260.184ASA grade 3–4 (n = 197)109 (55 %)51580.007Internal fixation69 (35 %)33360.420Hemiarthroplasty111 (56 %)37740.228Dynamic hip screw17 (9 %)7100.998Other1 (0.5 %)100.345No of. comorbidities:031 (16 %)328<0.001137 (19 %)7300.260252 (26 %)25270.005≥379 (40 %)44 (56 %)35 (29 %)0.001Complications during hospitalization:Model B.Pneumonia/chest infection8620.0010.0212.9161.175–7.237Urinary tract infection8238440.241Other infection3415190.528Wound infection000Deep wound infection1100.054Cardiac failure171250.0010.0102.4571.242–4.858Myocardial infarction6600.052Deep vein thrombosis1100.050Pulmonary embolism1100.0020.00133.2193.961–278.570Stroke3210.072Cancer3210.120Gastric ulcer7340.538Delirium12963700.0050.0182.0061.127–3.570Fallers3819190.267Number of falls7841370.262Fracture4220.609Luxation6330.416Reoperation8350.926Decubital ulcers3016140.044Combining baseline and complication factors:Model C.Age0.7051.0080.968–1.049Female0.1180.6550.386–1.113Dependence in P-ADL0.0072.3621.271–4.387Cancer<0.0013.3931.959–5.877Cardiovascular disease0.0132.0261.160–3.539Dementia0.0231.8831.091–3.250Pneumonia/chest infectionCardiac failure0.0182.2211.148–4.294Pulmonary embolism<0.00169.3967.107–677.632Delirium SD Standard deviation P-ADL Personal Activities of Daily Living ASA American Society of Anaesthesiologists classification HR Hazard ratio CI 95 % Confidence interval a p according to univariate cox regression model b p according to multivariate cox regression model The proportionality of hazards was tested using Schoenfeld residuals and time-dependent variables in extended Cox regression models [34]. A final multivariate model adjusted for age, sex and remaining significant variables from the two separate multivariate analyses was performed (Model C in Table 1). All calculations were carried out using SPSS v 23. All statistical tests were 2-tailed and a p-value <0.05 was considered statistically significant. BODY.RESULTS.PREDICTORS OF MORTALITY: A history of cardiovascular disease, cancer, dependence in P-ADL, dementia, having three or more co-morbidities, dependence in walking, having an ASA score of 3 or higher, living in residential care facilities, male gender, depression and pulmonary disease were all independently associated with time to death in univariate Cox regression analyses (Table 1). The results from multivariate Cox regression analyses are also displayed in the columns to the right of Table 1. The hazard ratios (HR) and 95 % confidence intervals (CI) are presented for the significant variables remaining in the multivariate models. When analysing associations between baseline variables and time to death we found that cancer, cardiovascular disease, dependence in P-ADL and dementia were associated with time to death in a Cox proportional hazard regression model adjusted for age and sex (Model A). In a second model (Model B.) complications during hospitalization were analysed; pulmonary emboli, pneumonia, cardiac failure and delirium were all associated with time to death. In the final model (Model C.) all significant variables in the two multivariate models above were combined; pulmonary emboli (HR 69.396, CI: 7.107–677.632), cancer (HR 3.393, CI: 1.959–5.877), cardiac failure (HR 2.221, CI: 1.148–4.294), cardiovascular disease (HR 2.026, CI: 1.160–3.539), dementia (HR 1.883, CI: 1.091–3.250) and dependence in P-ADL (HR 2.362, CI: 1.271–4.387) remained independently associated with all-cause mortality adjusted for age and gender. BODY.RESULTS.CO-MORBIDITIES AND BASELINE CHARACTERISTICS: The majority of the participants were women, living alone and independently; they walked independently indoors but only four out of ten were independent in P-ADL before the index hip fracture. Fifty-seven percent had a history of cardiovascular disease and 55 % had an ASA score of 3 or higher at baseline. The deceased had three or more co-morbidities in 56 % of the cases at baseline compared to 29 % among the survivors (Table 1). BODY.RESULTS.DEATHS: Seventy-nine out of 199 participants (40 %) died. Mortality was 13/199 (6 %) at 30 days and 34/199 (17 %) at 1 year after the index hip fracture. Twenty-six out of 51 (51 %) men died compared to 53/148 (36 %) women. After discharge, a total of 65 participants died during the 3 years of follow-up (Table 2).Table 2Primary causes of deathCause of deathDuring hospitalizationBetween discharge and 4 monthsBetween 4 and 12 monthsBetween 12 and 36 monthsTotalCardiovascular0121619Dementia0041418Hip fracture1130115Cancer023510Cerebrovascular00134Infection10012Deep infection00022Renal failure10102Fracture01102Gastrointestinal bleeding10001Ruptured aortic aneurysm00011Parkinson's disease00011Pancreatitis00011Gangrene01001Sum148124579Autopsy721415 BODY.RESULTS.CAUSES OF DEATH: In this study 13 participants died within 30 days of sustaining the index hip fracture. Ten of these 13 died during hospitalization: one before the operation, due to myocardial infarction; two on the day of operation, due to cardiovascular events; two the day after the operation (one due to gastrointestinal bleeding and one to myocardial infarction); one suffered a cerebrovascular event during the operation and never regained consciousness; one suffered a myocardial infarction after a week; two died of pneumonia and one died due to an intestinal infarction. The participants who died during hospitalization due to a cardiovascular event all had a history of cardiovascular disease. The three participants who died after discharge but within 30 days of the index hip fracture did so due to a cerebrovascular event, a myocardial infarction and a pulmonary emboli, respectively. The secondary cause of death within 30 days of the index fracture was due to a cardiovascular event in 6/13 (46 %) cases. In addition four participants died within 30 days after sustaining a new fracture. One of these participants fell and suffered a new hip fracture while still in hospital and then died due to an infection. After discharge one participant suffered a hip fracture and died due to pneumonia, one sustained a head trauma with a face fracture and died due to intracerebral bleeding and one had a knee fracture and died due to rupture of the aorta. Cardiovascular events (19/65), Dementia (18/65), cancer (10/65), fractures (6/65) and cerebrovascular events (4/65) were the most common primary causes of death after discharge (Table 2). BODY.RESULTS.COMPLICATIONS: In total 166 participants suffered from 583 infections, including 136 participants who suffered 363 urinary tract infections. Seventy-four participants suffered 111 cardiovascular events during the 3 years following the hip fracture. One-hundred and fourteen participants suffered 542 falls. Thirty-seven participants suffered 56 new fractures, including 13 new hip fractures, seven wrist fractures and seven rib fractures. Forty-nine participants suffered 60 decubital ulcers. During follow-up 96 participants had 234 hospital admissions with a total of 3984 days spent in hospital. Infections and cardiovascular events were more common after discharge among those who died (Table 3).Table 3Complications after femoral neck fracture occurring between discharge and until three year follow-up, differences between deceased and survivorsComplicationsBetween discharge and 4 months (n = 176)Between 4 and 12 months (n = 172)Between 12 and 36 months (n = 149)Pneumonia/chest infection58*21*Urinary tract infection51*5978Other infection344156Wound infection300Deep wound infection202Cardiac failure18*17*30*Myocardial infarction56*12*Deep vein thrombosis101Pulmonary embolism112Stroke2810Cancer044Gastric ulcer227Fallers536166Number of falls126130208Number of fractures61729Luxation324Reoperation327Decubital ulcers91011Number of participants hospitalized25*48*64Number of hospitalizations29*66*139*Number of days in hospital413*1134*2437*Median days in hospital, IQR9.0 (5.0–18.0)15.0 (4.0–31.2)23.5 (9.2–48.8)* = p < 0,05 according to Pearson's chi-square, Fisher's exact test or Mann–Whitney U test as appropriate, when comparing deceased and survivors IQR Inter Quartile Rate BODY.DISCUSSION: This study shows that both co-morbidities at baseline and complications during hospitalization are associated with mortality. Cancer, dependence in P-ADL, cardiovascular disease and dementia at baseline, and pulmonary emboli and cardiac failure during hospitalization were all independent predictors of mortality. Forty percent had died after 3 years despite the exclusion of those who were bedridden, had severe renal failure or pathological fractures. The most common primary causes of death were cardiovascular events, dementia, fractures, cancer and cerebrovascular events. The participants had several co-morbidities and suffered numerous complications such as infections, falls and fractures, cardiovascular events, delirium, and pressure ulcers. When analysing factors associated with death we found that both comorbidities and post-operative complications were of significance, a finding which is also verified in a recent review using data from a National Trauma Data Bank [35]. A study by Roche et al. [12] found that age, male sex, cancer, chest infection, cardiac failure and stroke could predict mortality but they did not include dementia or functional measurements in their model. Dementia, however, is common among hip fracture patients and was thus included in the present study. Dementia is also found to be a risk factor for death and in another large cohort study by Petersen et al. [19] age, cardiac complications and dementia were associated with mortality at 12 months but malignancies, cardiovascular disease and measurements of function were not included. In a large cohort study by Castronuovo et al. [36] heart disease was a risk factor for 30-days mortality but not during follow-up though no complications were included in the study. A reduced mental and medical status, and a poor physical ability at baseline were found among the deceased in the current study, similar to the results of a Norwegian study [23], although those living in nursing homes and patients who did not pass a mental status test had been excluded. Such patients were not excluded in the present study. Among those who died, 56 % had three or more co-morbidities at baseline and 65 % had an ASA score of 3 or higher. The number of co-morbidities and poor pre-fracture status might be indicators of frailty. It has been shown that the (ASA) classification of medical co-morbidities is strongly associated with medical problems in the perioperative period [37] and an earlier Swedish study shows that a high ASA score is a factor associated with mortality [21]. A cardiovascular disease is a strong predictor of post-operative cardiac failure, according to Roche et al. [12]. All participants in the present study who died during hospitalization due to a cardiovascular events had a pre-fracture cardiovascular disease. Early death within 30 days after admission to hospital due to a hip fracture has recently been described in a study that examines post-mortem reports, where respiratory infections and cardiovascular disease were the main causes of death [28]. These results are in line with earlier studies [24, 29] and our study shows a similar result as 46 % of early deaths were due to a cardiovascular event. During follow-up cardiovascular events, dementia and cancer were the most common causes of death in the present study, which is partly consistent with earlier studies [26, 27, 38]. The difference in the prevalence of infection as a cause might be due to the manner in which assessment of the causes of death was determined in the present study, as described above in the method section. There might also be an under diagnosis of dementia among many old people [39]. The participants in the present study were cognitively tested during hospitalization and at 4, 12 and 36 months. Our study confirms that complications among people suffering from a hip fracture are numerous, both early post-operatively and during follow-up. In-hospital complications among hip fracture patients, such as urinary tract infections, delirium, decubital ulcers and falls, can be successfully prevented and treated [14, 15] but as far as we know few intervention studies have succeeded in preventing cardiac complications [40–42]. Since we can now identify the most vulnerable patients, the focus in further research should be on prevention of infections and heart diseases in early postoperative care since they might be avoidable. These findings are in line with Petersen et al. [19] who concluded that cardiac complications constituted an important risk factor that might be modified and with Roche et al. [12] who emphasized the need for medical assessment among those with heart failure and chest infection. In addition, prevention should also focus on the numerous complications that occur after discharge from hospital. General complications were associated with loss of function in a recent study by Hansson et al. [13] Improved rehabilitation after stroke, including treatment of underlying comorbidities as well as secondary prevention, has increased the survival rate after stroke over the last few decades [43] while mortality after femoral neck fracture has remained constant. Treatment of risk factors for stroke and myocardial infarction as well as secondary prevention are currently well established in routine care. The 56 new fractures that occurred in the present sample indicate that fracture prevention also needs to become a part of routine care aimed at reducing fracture rates and mortality. Since hip fracture is, in many cases, an event that signals a systemic decline in the person's health, it is crucial to preserve and improve the clinical care pathways to ensure optimal recovery and survival for these patients. The present study will, hopefully, contribute to the knowledge available concerning the causes of death and highlight the potentially modifiable/preventable complications that we need to focus on in the future. The strength of our study is that we have systematically analysed all complications and the secondary causes of death among people with hip fracture, which no other study has reported to our knowledge. Differences in the present results compared to other studies might be due to the definitions applied to complications and co-morbidities and to the choice of exclusion criteria. In the future a more standardized description of the samples would facilitate comparison. In the present study we tried to obtain information about all events that the participants experienced and that led to death. An underlying cause of death is defined as "the disease or injury which initiated the train of morbid events leading directly to death, or the circumstances of the accident or violence which produced the fatal injury", in accordance with the rules of the ICD. Although the intention of the ICD is to provide a standard means of recording underlying causes of death, comparison of cause-of-death data over time and across countries should be undertaken with caution. The rules for selecting the underlying cause of death have been re-evaluated and sometimes changed. Incorrect or incomplete death certificates, misinterpretation of ICD rules for selection of the underlying cause, and variations in the use of coding categories for unknown and ill-defined causes might all occur, according to the WHO. There are some limitations to the present study. The sample was relatively small and the participants were only assessed three times over 3 years after discharge and there are certainly complications that were missed, despite the thorough reviews of the participants' medical records. As people with vertebral fractures and rib fractures do not always seek medical care such fractures are poorly documented. The number of vertebral and rib fractures has probably been underestimated in our study since x-rays were not routinely taken during follow-up. Suffering pulmonary emboli is a serious condition and the HR for pulmonary emboli in the multivariate analyse should be interpreted with precaution as only one person in this sample had an emboli and died soon after the fracture. BODY.CONCLUSION: Old people with femoral neck fracture have multiple co-morbidities and suffer numerous complications which might lead to death, both during hospitalization and after discharge. Thus randomized intervention studies should focus on prevention of complications that might be avoidable such as infections, heart diseases, falls and fractures. BODY.ABBREVIATIONS: ASA, American Society of Anaesthesiologists classification; CI, 95 % confidence intervals; DSM-IV, diagnostic and statistical manual of mental disorders, fourth edition; EpC, centre for epidemiology; HR, hazard ratios; ICD, International Classification of Diseases; P-ADL, personal activities of daily living; WHO, World Health Organization.

TITLE: Acute effect of Ethanol and Taurine on frontal cortex absolute beta power before and after exercise ABSTRACT: Ethanol (ET) is a substance that modulates the Central Nervous System (CNS). Frequently, ET intake occurs combined with energy drinks, which contain taurine (TA), an important amino acid found in the body (i.e brain and muscles). Although TA administration has been used in the improvement of physical performance, the impact of TA, ET and exercise remains unknown. This study aimed to analyze the acute effect of 6g of Taurine (TA), 0.6 mL∙kg-1 of Ethanol (ET), and Taurine combined with Ethanol (TA+ET) ingestion on the electrocortical activity before and after a moderate intensity exercise in 9 subjects, 5 women (counterbalanced experimental design). In each of the 4 treatments (Placebo—PL, TA, ET and TA+ET), electroencephalography (EEG) tests were conducted in order to analyze changes in absolute beta power (ABP) in the frontal lobe in 3 moments: baseline (before ingestion), peak (before exercise) and post-exercise. In the PL treatment, the frontal areas showed decrease in ABP after exercise. However, in the ET+TA treatment, ABP values were greater after exercise, except for Fp1. The ET treatment had no effect on the Superior Frontal Gyrus area (F3, Fz and F4) and ABP decreased after exercise in Fp1 and Fp2. In the TA treatment, ABP increased after exercise, while it decreased at the peak moment in most of the frontal regions, except for Fp1, F3 and Fz. We concluded that after a moderate intensity exercise, a decrease in cortical activity occurs in placebo treatment. Moreover, we found a inhibitory effect of TA on cortical activity before exercise and a increased in cortical activity after exercise. A small ET dose is not enough to alter ABP in all regions of the frontal cortex and, in combination with TA, it showed an increase in the frontal cortex activity at the post-exercise moment. BODY.INTRODUCTION: Ethanol (ET), or Ethyl Alcohol, is a substance widely consumed by population; however, its effects on the Central Nervous System (CNS) are not completely known yet. ET is able to alter the CNS excitatory and inhibitory neurotransmitter balance, most times favoring the inhibitory influence [1]. The acute effect of ET contributes to a decrease in cortical activity [2]. Given the importance of such substance, its influence in some regions of the cortex (i.e. the frontal cortex) has been widely studied. This area takes part in functions such as learning, planning, decision making, motivation, attention, working memory and sensory and motor information integration [3]. The frontal region is meant to monitor performance and to implement necessary adjustments in cognitive control [4]. According to reports from the literature, ethanol may cause changes in some functions, such as for example executive control, decision making and risk management [5]. In addition to changes in the CNS, ET acute effect may also harm other organism functions, such as the metabolic and cardiovascular ones, and it may therefore negatively affect physical performance [2]. Ethanol intake commonly occurs together with energy drinks, which contain an amino acid called taurine (TA), which objective is to reduce the symptons of depression [6]. Due to the different components contained in energy drinks, it is difficult to affirm the effects of each substance interacting with ethanol. While caffeine is considered to be an excitatory substance, TA may act as a CNS inhibitor, just like alcohol, through the stimulation of inhibitory receptor agonists [7]. TA has been used in the treatment of alcoholism and to improve physical performance. In animals, the quantity of extra-cellular TA in the CNS was found to increase immediately after alcohol ingestion, thus suggesting a protecting effect of cerebral activity deriving from alcohol intake [8]. Whereas the CNS has multiple and complex functions, the combination of these two related substances and the exercise could clarify physiological mechanisms in response to neural stimulus. Although TA has been used as an alternative therapy [9] and as a performance enhancer during exercise [10], no studies have been conducted with human beings about the acute effects of such amino acid on electrocortical activity in the frontal cortex, when ingested alone or together with alcohol, before and after exercise. Quantitative electroencephalography (qEEG) is a non-invasive tool of electrocortical activity analysis, and it has been widely used to assess the influence of physical exercise and of different substances in the central nervous system. After exercise, cortical activity may increase or decrease, depending on the physical exercise protocol and on the analyzed cortical region [11]. Beta frequency band has been used to evaluate the post-exercise effects, mainly in the brain frontal regions [11–13]. Nevertheless, the impact of the TA, ET and exercise on EEG activity remains unknown. Our objective was to apply the qEEG in order to investigate the acute effect of ET, TA and both of these substances together in the frontal cortex before and after exercise. In particular, beta absolute power (13–30 Hz) was analyzed; this is a fast frequency and it is associated with the alertness/attention state and with motor activity. We hypothesize that the cortical activity (ABP) may be affected by these substances in the frontal cortex areas before and after exercise. Furthermore, TA may modulate ET effect when ingested together. BODY.MATERIALS AND METHODS.SUBJECTS: All participants, five healthy women (22 ± 3 years) and four healthy men (26 ± 5 years), were right-handed, regular drinkers of alcoholic beverages, amateur practitioners of physical exercise and non-smokers. The participants were asked to avoid physical activities with more than 5 metabolic equivalents (METs) and any food containing caffeine, alcohol and taurine for 48 hours before the test. Individuals with liver disorders(as determined by the activity of the enzymes aspartate amino-transferase, alanine aminotransferase, direct and indirect bilirubin, alkaline phosphatase, gamma-glutamyl transferase and lactatedehydrogenase) or who were taking medications, as well as alcohol users with a weekly intake greater than fifteen or less than two alcohol servings were excluded from this study. Volunteers read and signed an informed consent form. All experimental procedures were approved by the local ethics committee of University Hospital Clementino Fraga Filho (HUCFF) at Federal University of Rio de Janeiro (03899312.5.0000.5257). All of research protocols described here were carried out in accordance to the declaration of Helsinki". BODY.MATERIALS AND METHODS.EXPERIMENTAL DESIGN: This study lasted approximately five weeks for each participant. The experiment began with an anthropometric and an effort test. During the following visits (once a week), a particular intervention was applied in randomized order (PL, TA, ET or TA+ET). The experimental design of these tests was in agreement with a specific treatment, using a protocol described previously [14]. Six grams of microcrystalline cellulose were diluted in 150 ml of an artificial orange juice (Clight®, 21 g.L-1) and used as the placebo (PL) solution, while six grams of powdered taurine (TA) were used for the experimental solution. Ethanol (Orloff® vodka, Resende, RJ, Brazil—38% alcohol content) was administered in doses of 0.6 ml. kg-1, combined with the ingestion of artificial orange juice in a proportion of 2:1 (juice: vodka). The TA and PL solutions were ingested two hours before the exercise, while the ET alcoholic beverage was ingested thirty minutes before the exercise. Such ingestion time was determined according to the peak plasma levels of each substance after intake [15]. In the TA+ET treatment, ethanol was ingested one hour and thirty minutes after the administration of taurine; Taurine administration followed a simple double-blind procedure. The EEG was recorded at the peak and post-exercise. Since the frontal region is related to executive functions, our aim was to observe the frontal regions dynamics in order to evaluate cognitive alterations when subjects were under drug influence. BODY.MATERIALS AND METHODS.ERGOMETRIC PROTOCOL: A continuous and maximal load ramp testing on a cycle ergometer with an electromagnetic brake (Imbrasport®, Porto Alegre, RS, Brazil) was used to determine maximal power output (Wmax), maximal aerobic power (V ̇O2max) and anaerobic threshold (Vista Mini-CPX®, Vacumed®, Ventura, CA, USA). The test started with the subject seated on a cycle ergometer for six minutes, followed by a four-minute warm-up pedaling without any load. After the warm-up, the maximum test was performed according to the procedures described by Nogueira and Pompeu [16]. The effort perceived exertion was determined at the end of each minute. A constant-load protocol (SWT), with an intensity 10% lower than the load of the anaerobic threshold set for the maximum effort test was adopted for 10 minutes at a rate of 60 revolutions per minute (RPM) as a physical tool for the ergometric test, causing a stress on the body, in order to help in the analysis of ABP under the influence of PL, TA, ET and TA+ET. BODY.MATERIALS AND METHODS.BLOOD COLLECTION—BIOCHEMICAL ANALYSIS: Serum and plasma were obtained from peripheral blood samples at three moments (at the beginning, thirty minutes after ethanol ingestion and immediately after exercise) for each treatment (ET and TA+ET). Serum levels of liver enzymes were determined using the VITROS® Chemical System and plasma Ethanol levels by Siemens Dimension® Series. BODY.MATERIALS AND METHODS.ACQUISITION OF ELECTROENCEPHALOGRAPHIC SIGNAL: The EEG signal capture was performed using the BrainNet-BNT device 36 (EMSA, Medical Instruments, Brazil). Twenty monopolar electrodes were arranged following the 10/20 International System Protocol. The impedance of the electrodes was maintained between 5 and 10 kΩ. Recorded data had a total range of less than 70 μV. The data signal was amplified with a gain of 22,000, analogically filtered between 0.01 Hz (high-pass) and 80 Hz (low-pass), and sampled at 200 Hz. The Data Acquisition software (Delphi 5,0TM, USA) from the Brain Mapping and Sensory Motor Integration Lab was employed with the digital notch filter (60 Hz). BODY.MATERIALS AND METHODS.DATA PROCESSING AND ANALYSIS: Visual inspection and independent component analysis (ICA) were applied to remove possible sources of artifacts produced during data collection. The data were collected using a bi-auricular reference, and they were transformed using the average reference after ICA was applied and artifact elimination was concluded. Through ICA and visual inspection, all the ranges which clearly showed artifacts such as blinking and muscle-related movements were removed. A classic estimator was applied for the power spectral density (PSD) performed by MATLAB 5.3 (Matworks, Inc.). BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Statistical analysis was performed using the Statistical Package for the Social Sciences® (SPSS® Inc., Chicago, IL, USA), SigmaPlot® (Systat® Software Inc., Chicago IL, USA) and Microsoft Excel® for Windows® (Microsoft®, Redmond, WA, USA). In order to analyze the absolute power, two-way ANOVA was used (treatment vs. moment) in order to assess the interaction of absolute beta power (ABP) between treatment and moment for all frontal electrodes. When an interaction between factors was found, a one-way ANOVA (Bonferroni post-hoc) was applied to each treatment for all frontal electrodes. A ANOVA one-way was conducted to compare ethanol concentration in the plasma between moments (baseline, peak and post-exercise) to each treatment (ET and TA+ET). A Student t-test was used to compare ethanol concentration in the plasma moments in different treatments (ET Baseline vs. TA+ET Baseline; ET peak vs. TA+ET peak; ET post-exercise vs. TA+ET post-exercise). Descriptive statistics were used with the mean ± standard error (se) (p ≤ 0.05). The p-value was adjusted through the bonferroni correction for multiple comparisons. The effect size was estimated using the Cohen's d method. BODY.RESULTS: When investigating the ethanol concentration in the plasma in ET and TA+ET treatments, a significant increase was seen for both treatments, compared to the Baseline values (0 mmol.L-1). However, there was no significant difference between baseline, peak and post-exercise to each treatment. Also, there was no significant difference between treatments when comparing the specific moments (ET Baseline vs. TA+ET Baseline; ET peak vs. TA+ET peak; ET post-exercise vs. TA+ET post-exercise) (Table 1). 10.1371/journal.pone.0194264.t001 Table 1 Plasma ethanol concentrations. Treatment Plasma ethanol concentration (mmol . L -1 ) 30 min after the ingestion P value Post-exercise P- value TA+ET 13.7 ± 2.9 ** 0.001 10.9 ± 1.4 ** 0.001 ET 12.3 ± 4.3 ** 0.001 9.7 ± 2.0 ** 0.001 Values are expressed as means and SDs. Baseline values equal to 0 mmol . L -1 . ** Significant difference compared to the baseline for each treatment. A two-way ANOVA (moment vs. treatment) demonstrated interaction between the two factors for all investigated electrodes [Fp1- F(11;13795) = 7.156, η2p = 0,003, p<0.001; Fp2—F(11;13795) = 17.794, η2p = 0,008, p<0.001; F7—F(11;13795) = 26.174, η2p = 0,011; F8—F(11,13795) = 29.548., η2p = 0,013, p<0.001; F3—F(11,13795) = 22.903, η2p = 0,010, p<0.001; Fz—F(11,13795) = 18.800, η2p = 0,008, p<0.001; F4—F(11,13795) = 26.289, η2p = 0,011, p<0.001]. With the aim to analyze in detail all interactions found between moment and treatment, a one-way ANOVA was conducted separately for each treatment (PL, TA, ET and TA+ET). Interaction analyses are reported below. The effect sizes for all variables were summarized in S1 Table. BODY.RESULTS.ANTERIOR PREFRONTAL CORTEX (FP1 AND FP2): Difference was found at Fp1 between the baseline and post-exercise moments (p<0.05) and between peak and post-exercise (p<0.05) for the PL, ET and TA treatments. On the other hand, at Fp2, difference was found among moments for all treatments. For the PL, ET and TA treatments, difference was observed among all moments (p<0.05). For ET+TA, significant difference occurred between the baseline and post-exercise (p<0.001) and between the peak and post-exercise (p = 0.009) moments (Fig 1). 10.1371/journal.pone.0194264.g001Fig 1Absolute beta power of anterior prefrontal.Values are expressed as means and standard error. * Significant difference (p ≤ 0.05). This figure shows the absolute beta power of anterior prefrontal area (Fp1 e Fp2), in each treatment (Placebo, Taurine, Ethanol and Taurine + Ethanol) before (baseline and peak) and after (post-exercise) exercise. BODY.RESULTS.INFERIOR PREFRONTAL GYRUS (F7 AND F8): For the F7 electrode, difference was found between the baseline and peak (p<0.05), and between peak and post-exercise (p<0.05) moments for the ET and TA treatments. For the TA+ET treatment, difference occurred between the baseline and peak moments (p<0.001), and between baseline and post-exercise (p = 0.021). On the other hand, in the PL treatment, difference was observed between the baseline and post-exercise moments (p<0.001), and between peak and post-exercise (p<0.001). At F8, significant difference was found between the baseline and post-exercise moments (p<0.05) and between peak and post-exercise (p<0.05) for the ET and ET+TA treatments. For the PL treatment, significant difference was identified between the baseline and peak moments (p<0.001) and between baseline and post-exercise (p<0.001). And for the TA treatment, difference was found at all moments (p<0.05) (Fig 2). 10.1371/journal.pone.0194264.g002Fig 2Absolute beta power of inferior prefrontal gyrus.Values are expressed as means and standard error. * Significant difference (p ≤ 0.05). This figure shows the absolute beta power of inferior prefrontal gyrus (F7 e F8), in each treatment (Placebo, Taurine, Ethanol and Taurine + Ethanol) before (baseline and peak) and after (post-exercise) exercise. BODY.RESULTS.SUPERIOR FRONTAL GYRUS (F3, FZ AND F4): For the F3 electrode, significant difference was only found for PL and ET+TA. In the PL treatment, significant difference was observed between all moments (p<0.05); in the ET+TA treatment, difference occurred between the baseline and peak moments (p<0.001) and baseline and post-exercise (p<0.001). For the F4 electrode, significant difference was found among all moments in the TA treatment (p<0.05). In the ET+TA treatment, significant difference was found between the baseline and post-exercise moments (p<0.001), and between baseline and peak (p<0.001). On the contrary, for the PL treatment, difference was observed between the baseline and post-exercise moments (p<0.001) and between peak and post-exercise (p<0.001). At Fz, just like at F4, significant difference occurred among all moments (p<0.05) for the TA treatment. Significant difference was found between the baseline and post-exercise moments (p<0.001) and between baseline and peak (p<0.001) for the ET+TA treatment. In the PL treatment, significant difference was observed between the baseline and post-exercise moments (p<0.001), and between peak and post-exercise (p<0.001) (Fig 3). 10.1371/journal.pone.0194264.g003Fig 3Absolute beta power of superior frontal gyrus.Values are expressed as means and standard error. * Significant difference (p ≤ 0.05). This figure shows the absolute beta power of superior prefrontal gyrus (F3, F4 e Fz), in each treatment (Placebo, Taurine, Ethanol and Taurine + Ethanol), before (baseline and peak) and after (post-exercise) exercise. BODY.DISCUSSION: The present study aimed to analyze the effect of TA, ET and these two substances administered together on the electrocortical activity, before and after moderate intensity exercise. An overwiew of these results was available as a cartoon (Fig 4). Specifically, changes in the absolute beta power in the frontal cortex were assessed. We hypothesize that the cortical activity (ABP) may be affected by these substances in the frontal cortex areas before and after exercise. Furthermore, TA may modulate ET effect when ingested together. Despite acknowledging the complexity of cerebral activity and of its multiple networks at various frequencies, due to methodological reasons, the discussion of electrophysiological data has been divided into three different subtopics, related to the areas associated with the investigated electrodes, as follows: Superior Frontal Gyrus—SFG (F3, Fz and F4); Anterior Pre-Frontal Cortex—PFC (Fp1 and Fp2); Inferior Frontal Gyrus–IFG (F7 and F8). 10.1371/journal.pone.0194264.g004Fig 4A cartoon representation demonstrates an overview of the absolute beta power results in different areas of frontal cortex.The arrow "↑" means an increase of absolute beta power. The arrow "↓" means a decrease of absolute beta power and the double arrow "↔" means no modification in absolute beta power. BODY.DISCUSSION.SUPERIOR FRONTAL GYRUS—SFG (F3, FZ AND F4) AREA—8 AND 8L: Brodmann area 8 is considered to be an important area for motor learning. Studies from the literature have demonstrated this area to be responsible for starting, keeping, coordinating and planning complex movement sequences [17]. The transformation of such sensory signals into motor responses occurs through the visual and auditory systems [18]. In addition, this area also acts in working memory [19] and it may be involved in a cognitive control process, which is able to modulate the current emotional state [20]. As expected, no difference was found between the baseline and peak moments in the PL treatment for most of the electrodes relate to this region (i.e., Fz and F4), except in F3. However, a decrease in beta power was observed at the post-exercise moment, when compared to the others. Kubitz and Pothakos [21] have also observed a decrease in beta values after exercise. Nevertheless, Moraes et al. [12] and Bailey et al. [22] verified an increase in absolute beta power for F3 and F4 immediately after exercise. Kubitz and Mott [23] have not found any difference in absolute beta power before or after exercise. The different exercise intensity and duration and the time of cortical activity measurement may be involved with the different results. Such difference in the methods leads to different physiological responses (i.e. temperature, cerebral blood flow and hypoxia), which may influence the brain, therefore generating different results Lardon and Polich [24]. Bailey et al. [22] state that exercise intensity may affect cerebral activity: the greater the intensity, the greater the cerebral activity and, therefore, the beta values. The time of cortical activity measurement after exercise may also influence on beta values; in this same study, such values were found to be greater immediately after exercise, when compared to the pre-exercise moment, showing no difference between the pre- and post- moments when measured 10 minutes after exercise. Results by Petruzzello and Landers [25] also show a decrease in cortical activity along time for F4, when a comparison was done between the 10th and 30th minute after exercise. In the TA treatment, difference was found for the Fz and F4 electrodes, with TA causing beta increase at Fz and beta decrease at F4. With regards to the post-exercise moment, an increase in beta power was found for both electrodes, when comparing this moment to the others. Taurine is used to improve exercise performance. Reports from the literature highlight some metabolic benefits for the membrane, fat oxidation, insulin sensitivity and cardiac muscle; however, its acute and chronic effects on the CNS need to be better investigated during exercise [10, 15] (Balshaw et al., 2013; Galloway et al., 2008). Taurine ingestion may benefit the cardiac and skeletal muscles, by modulating the Calcium channels, therefore favoring cardiac contraction and helping with blood distribution [7]. Such greater cerebral blood flow, deriving from the response to TA ingestion, may influence greater cerebral activation (greater ABP values) at FZ and F4, after exercise [24]. It is important to highlight that no significant difference was found in the ET treatment for this region. Such findings are in agreement with the results in the studies by Ehlers et al. [26] and Cohen et al. [27], where no significant difference in ABP was found for the F3 and F4 electrodes (left and right side of this area, respectively), when comparing the moments before and after substance intake. Such responses may have occurred due to the similar ET quantity ingested in both studies mentioned in this research, with a consumption level between 0.6 and 0.8 ml/kg. In spite of such result, Zheng et al. [28] found ET to have an effect on the superior frontal gyrus, pointing out possible alterations in the CNS, such as: memory, motor control, cognitive ability and spatial function. The aforementioned authors observed changes in the alpha band values (lower frequencies) in this region, thus justifying the ET depressive effect in this area. On the other hand, in the ET+TA treatment, similar results were found for the whole superior frontal gyrus region (F3, F4 and Fz), with an increase in ABP at the peak and exercise moments, when compared to the baseline moment. Since no changes in ABP were found in this region for the ET treatment, and since similar results were found for the TA treatment after exercise, TA is suggested to stand out, with similar physiological adaptations to the TA treatment during exercise, thus influencing beta values increase after exercise. No reports about the effect of both TA and TA+ET on this region are present in the literature. Network changes in this region may cause impairment in motor and executive functions, working memory and high-demand cognitive functions [29]. Li et al. [30] observed a decrease in the grey matter volume in the SFG in patients with Parkinson's disease, when compared to healthy individuals, suggesting a relation with the motor impairment typical of the disease. The greater ABP values found after exercise, in both TA and TA+ET treatments, when compared to baseline indicate greater cerebral activity after exercise in this region, with possible function improvement. BODY.DISCUSSION.ANTERIOR PRE-FRONTAL CORTEX (FP1 AND FP2): The pre-frontal cortex (Brodmann area 10) has been associated with complex executive functions (executive control of behavior, decision making and inferential reasoning) and it plays an important role in memory control and manipulation, in addition to being also related to emotional and cognitive tasks [31]. On the other hand, beta is a fast frequency associated with attention processes, arousal and movement execution [32]. Our results highlighted an interaction between moment and treatment factors, that is, changes in the beta cortical activity in this region will occur according to the ingested substance and to the treatment moment (i.e., baseline, peak or post-exercise). Specifically, when investigating the left anterior pre-frontal cortex (Fp1) and the right pre-frontal cortex (Fp2), the TA and ET conditions were found to have an effect on the right pre-frontal cortex during the performance peak, with no difference between the baseline and peak moments in the left pre-frontal cortex for all conditions. In particular, all substances caused a decrease in beta power during its performance peak. As observed for ABP, changes in the right hemisphere may be related to the substance effects on some specific functions of this area. The anterior pre-frontal cortex is directly related to visuospatial prospective memory. In addition, the ABP oscillations are associated with motor activity, thus we hypothesized this frequency band to be involved in keeping the current sensory/cognitive state [32]. This region is strictly related to risk decision making; according to Kwon et al. [31], risk decisions are more related to the right side of this region, suggesting these decisions to be dependent on the level of activation of this side. On the other hand, Aron et al. [33] state the right side to be responsible for suppressing memories and for responding to visual and auditory distractions. According to Arnsten [34], the PFC is crucial to centralize attention onto a specific task and to regulate behavior and emotions, especially to inhibit inappropriate emotions, impulses and habits. This region is also necessary for goal planning as well as behavior and thought organization. Mainly the right hemisphere is important to regulate distractions, inappropriate behavior and emotional response. As with regards to specific treatments, decrease in ABP was found in the PL treatment for the post-exercise moment, when compared to the baseline moment. The different intensities and duration of exercise have an influence in the acute physiological adaptations, which could interfere in the electrocortical activity. For example, catecholamines and temperature regulation, also modulated by the hypothalamic nucleus, may influence beta activity [35]. However, more studies are needed to better understand the mechanisms inherent to the relation between different exercise protocols and beta activity in this specific region. When analyzing the ET+TA combination, a significant effect was found only in the right anterior pre-frontal cortex, with ABP increase at the post- moment, when compared to the others. Our results suggest the intake of the ET+TA combination alone do not provoke any changes in the anterior pre-frontal cortex; however, exercise potentializes the combination of these two substances, thus causing significant beta power increase in the right anterior pre-frontal cortex. As previously said, this area is directly related to executive control, specifically motor and behavioral executive control, attention, and inhibitory control. In particular, the right hemisphere features characteristics related to spatiality and development of direction sense. Therefore, this study hypothesized beta power increase at Fp2 to be associated with an increase in attention and executive motor control, required for the performance of physical exercise. Despite a beta decrease at the peak moment, TA provokes an increase in beta power during the exercise moment in both hemispheres of the anterior pre-frontal cortex, when ingested alone. Therefore, depending on the region, taurine may decrease electrocortical activity; this finding is in agreement with other studies demonstrating the inhibitory effect of this amino-acid on the central nervous system [36]. However, the acute effect of taurine seems to be related to the effect of exercise, with an increase in beta activity after exercise. Some studies found taurine to also increase glutamate receptor activity, through the regulation of cytoplasmic and intramitochondrial calcium regulation [37]. Even when taurine is ingested with alcohol, ABP increased after exercise for Fp2. Since beta plays an initial role in the selective inhibition of some actions and in cognitive control [38], it is possible to suggest that TA may have two important effects: first, it may diminish beta activity and negatively alter the two aforementioned tasks; secondly, it may increase cortical activity and, consequently, beta values, thus improving the performance of such tasks. In the ET treatment, this substance was found to also play an important role in the right anterior pre-frontal cortex; however, in addition to the ET intake alone decreasing ABP, the combination of ET and exercise also provokes decrease in beta power. Such results are in agreement with the literature about ethanol effects before and after intake, with lower cortical activity caused by the acute effect of the substance [39] mainly at Fp2 [40]. Such electrocortical activity decrease in this region may negatively influence some processes, such as working memory and decision making [5]. This may happen due to the ethanol effects on the CNS, directly facilitating GABA neurotransmitter transmission and inhibiting the glutamate neurotransmitter functions [1]. Few studies have related beta band to this region (PFC). Researches in the literature performed with animals (monkeys) affirm that beta neuronal synchronization involves execution tasks, such as rule selection. A given rule is represented by a group of PFC neurons, oscillating in synchrony at this frequency [41]. Buschman et al. [42] also demonstrate beta band synchrony in the PFC of monkeys to select the most relevant group of rules. Studies with humans highlight beta oscillations to start the cognitive control and selective inhibition process in the PFC [38]. In other words, specifically in this region, the alcohol effects are found and suggested to significantly influence inhibition decrease. Empirically, it is possible to observe people who consume alcohol in bars and restaurants becoming more extroverted and disinhibited and acting in a certain way, only because they were under the influence of such substance. BODY.DISCUSSION.INFERIOR FRONTAL GYRUS–IFG (F7 AND F8): Traditionally, the IFG has been widely associated with aspects related to language and semantics [43]. In addition to this, the activation of such area may be related to selection tasks and working memory (spatial and non-spatial) [19]. Activities in this area may also reflect the inhibition of a motor act associated with a reactive emotional response [20]. Increase in beta activity may be associated with increased cortical activity, and according to Gilbertson et al. [44], beta increase may be related to worse movement performance, demonstrating triggered voluntary movements to be smaller when beta activity is greater. However, the ability to perform a Go/NoGo task was associated with the increase in beta band activity in the IFG [45]. In this region, interaction between the moment and treatment factors was also found for the two analyzed electrodes. In the PL treatment, decrease in ABP occurred at the baseline moment, compared to the post-exercise moment in this area. Schneider et al. [13] have also observed similar results after moderately intense running, that is, exercise causes decrease in absolute beta power. A study by Li et al. [11] is in agreement with such results; they identified a decrease in the left IFG cortical activity after exercise. On the other hand, in the ET+TA treatment, increase in ABP was found for the peak moment, when compared to baseline only at F7, demonstrating this treatment to influence beta power only in the left IFG. However, beta increase was verified at the post-exercise moment for both left and right IFG, showing an influence of exercise on absolute beta power in both hemispheres. Despite acamprosate (a substance containing TA) being used in the treatment of alcoholics in order to reduce ethanol-abstinence symptoms Plosker [46], there are no studies in the literature about the acute effect of TA+ET on the electrocortical activity in humans. Taurine seems to maximize the effects of alcohol in the left hemisphere of the IGF, considering that beta activity stayed high after exercise in the TA+ET treatment. On the other hand, in the right hemisphere, the TA effect together with ET did not modify beta activity, considering that the TA+ET results were similar to the ET ones. In great part of the frontal cortex, TA seems to increase beta activity after exercise, even with ET intake. We hypothesized the increase in cortical activity after exercise to be related to the TA effects on the modulation of the glutamate receptor activation, through the cytoplasmic and intramitochondrial calcium regulation [47]. In a study with animals, the acute administration of TA+ET, TA was proved to exert a neuroprotecting action on the apoptosis induced by the acute effect of ethanol on the cerebellum [48]. The pioneer present study allows verifying the cortical activity of the TA+ET acute administration in humans, but the mechanisms inherent to the effects of such substances on the CNS need to be furtherly studied. In the ET treatment, a substance influence on the left hemisphere of this area (F7) was found, provoking an increase in beta power. However, when ET is combined with exercise, an increase in beta power is found in the right hemisphere (F8), while decrease in beta power is found in the left hemisphere (F7) of this region. The area correspondent to the right hemisphere (F8) is activated when cognitive tasks require inhibition [49]. In addition to the function related to language, the IFG activation may occur during the execution of distal movements, such as grabbing, and is also directly related to mirror neurons for expressive movements [50]. We suggest that some of these specific functions related to the IGF may have been influenced by alcohol intake and exercise, seen through changes in beta activity. Therefore, it is possible to observe this small dose of ET to alter the left IFG activity, thus possibly affecting some specific functions of this region, such as attention [19]. In terms of TA treatment, TA was seen to provoke a decrease in beta power in both hemispheres. Furthermore, in TA treatment, absolute beta power increased in the right side of this region after exercise, thus showing interaction between such amino acid and exercise. Such effect may interfere in some tasks associated with this region, since the right inferior frontal gyrus is directly related to some specific brain functions, such as inhibitory and attention control [51]. The absolute beta power decrease at the peak moment may be explained by its already proven inhibitory effects [47]. Studies involving the effect of TA have been increasing in recent years due to their potential physiological influence. In addition, the simultaneous use of TA and ET justifies a better understanding of the acute effects of these substances. The complexity of CNS and the multiple, distinct, and important functions that each of its regions play, generate innumerable interpretations of brain functions. Thus, the combination of two substances that appear to be metabolically associated, and exercise as a physiological stressor, provide a more comprehensive understanding of the relationship of those compounds and the operation of CNS. Although our work has shown attractive results, some limitations could be highlighted: the amount of ET administered in comparison to some studies was small. It would be applicable to evaluate these effects in higher ET concentration. The taurine peak levels were defined from literature data. The analysis of taurine serum levels could avoid possible problems involving individual variability of this measure and also improve the explanation of the results. It is a fact that counterbalanced design favors the number of cell cases for each condition and, consequently, the sample size. However, evaluating a larger number of subjects and the gender effect would be interesting due to the great interindividual variability of neural measures. In summary, due to many different results, we can highlight that the acute effect of placebo ingestion decreased the electrocortical activity after exercise. The TA acute effect seems to inhibit the superior frontal region at the plasma peak; however, in such region, the TA effect increases ABP after exercise. A small ET dose was not enough to alter beta activity in the superior frontal area and it was sufficient to diminish it on the left side of the brain (Fp1 and F7) after exercise. We could observe that the ingestion of taurine in combination with ethanol showed different results of the ingestion of ethanol separately. TA when ingested together with ET had an similar effect in the right side (Fp2, F8, F4) of the frontal cortex that TA treatment after exercise. More research is thus needed to better understand the mechanisms involved in the CNS response, such as electrocortical activities and frontal cortex functions, to such substances (TA and ET) with different dosage, exercise type and intensity. BODY.SUPPORTING INFORMATION: S1 TableThe effect sizes for variables of frontal cortex, moments and treatments.(DOCX)Click here for additional data file.

TITLE: Sensitivity to scale of willingness‐to‐pay within the context of menorrhagia ABSTRACT.ABSTRACT.OBJECTIVES: Willingness‐to‐pay (WTP) provides a broad assessment of well‐being, capturing benefits beyond health. However, the validity of the approach has been questioned and the evidence relating to the sensitivity of WTP to changes in health status is mixed. Using menorrhagia (heavy menstrual bleeding) as a case study, this exploratory study assesses the sensitivity to scale of WTP to change in health status as measured by a condition‐specific measure, MMAS, which includes both health and non‐health benefits. The relationship between EQ‐5D and change in health status is also assessed. ABSTRACT.ABSTRACT.METHODS: Baseline EQ‐5D and MMAS values were collected from women taking part in a randomized controlled trial for pharmaceutical treatment of menorrhagia. Following treatment, these measures were administered along with a WTP exercise. The relationship between the measures was assessed using Spearman's correlation analysis, and the sensitivity to scale of WTP was measured by identifying differences in WTP alongside differences in MMAS and EQ5D values. ABSTRACT.ABSTRACT.RESULTS: Our exploratory findings indicated that WTP, and not EQ‐5D, was significantly positively correlated with change in MMAS, providing some evidence for convergent validity. These findings suggest that WTP is capturing the non‐health benefits within the MMAS measure. Mean WTP also increased with percentage improvements in MMAS, suggesting sensitivity to scale. ABSTRACT.ABSTRACT.CONCLUSION: When compared to quality of life measured using the condition‐specific MMAS measure, the convergent validity and sensitivity to scale of WTP is indicated. The findings suggest that WTP is more sensitive to change in MMAS, than with EQ‐5D. BODY.INTRODUCTION: Contingent valuation is a method for assigning monetary values to non‐market goods, such as health‐care interventions, for use as an outcome measure within cost–benefit analysis (CBA). Willingness‐to‐pay (WTP) is the most commonly used contingent valuation approach and provides an overall measure of strength of preference expressed in monetary terms. Using this approach, individuals are asked to consider hypothetical scenarios that describe both the process and outcome of the health‐care intervention and asked to state maximum WTP for the health care good being valued. Sample average WTP values are typically used as an indication of strength of preference and can be directly compared to assess the value of alternative health‐care interventions. In a similar way to how EQ‐5D is used to inform the outcome measure for cost–utility analysis (CUA), WTP can also be used to inform the outcome for a CBA. Used in this way, it becomes a generic measure of value for treatment or services and is thus weighed against cost to measure overall cost‐effectiveness. Depending on the ratio between the incremental difference in costs and benefits (WTP) of alternative treatment options, judgements can then be made by decision makers on whether to recommend the treatment/service based on this ratio. WTP encapsulates both health and non‐health aspects of well‐being, the advantages of such an approach are ever more recognized, particularly because the National Institute for Health and Care Excellence are now also commissioning across public health and social care.1 Despite this, there are key limitations to the WTP approach including the difficulty with contemplating the hypothetical survey scenario, and the lack of well‐defined preferences when individuals are unfamiliar with the goods they are asked to value. The literature also refers to evidence on strategic bias and questions about the validity of the approach.2 The objective of this exploratory study was to examine the validity of the WTP approach using treatment for heavy menstrual bleeding (clinically termed menorrhagia) as a case example. Menorrhagia is a chronic condition with episodic symptoms which is known to affect both health and non‐health aspects of life. Generic quality of life measures, such as EQ‐5D and SF‐6D, that are focused on health alone are recommended for use in health care to be used to capture the impact of conditions. The advantage of these generic measures is that they enable comparison of effectiveness across different treatment conditions as the outcomes are measured using one commensurate unit. An interesting feature of menorrhagia is the condition's chronic but episodic nature as the symptoms occur for approximately 1 week every month which has implications for the timing of assessment when using generic measures with standard recall periods.3 In terms of validity, within the context of menorrhagia the condition‐specific quality of life measure, MMAS (menorrhagia multi‐attribute scale described in detail below), is considered to be the gold standard measure.4 Whilst condition‐specific measures can be more sensitive than generic measures, their use in decision making is limited due to the lack of comparability across conditions. There are several types of validity that one can assess including content validity (whether all relevant aspects of the condition are considered in the instrument), construct validity (determines whether an underlying relationship exists between questions in the instrument and an attribute that is measured), and criterion validity (whether one attribute or set of attributes predicts an outcome based on information from other attributes).5, 6 The focus of this paper is construct validity, or more specifically convergent validity which is the degree to which two theoretically equivalent measures converge, and within the context of contingent valuation, it is often referred to as sensitivity to scale or scope.7 This question is of theoretical relevance as many studies have shown that WTP demonstrates theoretical validity with WTP increasing with income,8 and others have focused on convergent validity with other preference elicitation measures such as time trade‐off (TTO) and standard gamble (SG).9 The evidence on convergent validity within the health‐care sector and sensitivity to the size of the benefit is mixed 9, 10 and in particular, the evidence relating to the sensitivity of WTP to changes in health status 9 is far from conclusive. In theory, the respondent's perception of the value of a treatment should be sensitive to changes in the size of the benefit derived from the treatment. For example, WTP demonstrates convergent validity if the WTP increases with a perception of greater improvement in treatment benefit. This study presented a unique opportunity to assess the sensitivity of WTP longitudinally as both EQ5D and MMAS data were collected at different time points, along with WTP. The sensitivity to scale of WTP can therefore be assessed in relation to EQ5D and MMAS over time. First, we assessed the change in condition‐specific quality of life following treatment for menorrhagia, as measured by MMAS, against the WTP for this change in outcome, and second, we assessed the underlying relationship between WTP and general health‐related quality of life when measured using EQ‐5D. BODY.METHODS: We carried out the exploratory study with women who were already participating in the NIHR funded ECLIPSE trial (ISRCTN86566246).11 Ethical approval was obtained from the National Research Ethics Service Committee South West‐Exeter and clinical trial authorization from the Medicines and Healthcare Regulatory Authority. Written consent was obtained from the participants. BODY.METHODS.STUDY POPULATION: The ECLIPSE trial is a pragmatic, multicentre, randomized trial, comparing the clinical and cost‐effectiveness of levonorgestrel‐releasing intrauterine device (LNG‐IUS) against usual medical treatment in the primary care setting.11, 12 Women between 25 and 50 years of age that presented to their general practitioner (GP) with menorrhagia, occurring over at least three consecutive cycles, were randomized to a treatment group by telephone or web‐based central randomization service. Women were excluded if they intended to become pregnant over the next 5 years, were taking hormone replacement therapy or tamoxifen, had intermenstrual or post‐coital bleeding or examination suggestive of fibroids (abdominally palpable uterus equivalent in size to 10–12 weeks of gestation) or other pathologies, or had contraindications to, or a preference for, LNG‐IUS or usual medical treatments. The pharmaceutical treatments were either the LNG‐IUS (termed Mirena in the questionnaire) or usual medical treatment (oral treatment), which can include tranexamic acid, mefenamic acid, combined oral contraceptive or Depo‐Provera. BODY.METHODS.DATA COLLECTION: Data were collected at baseline and once symptoms had stabilized with treatment ('post‐initial treatment effectiveness'). As the study is nested within the ECLIPSE trial, ECLIPSE trial data collection forms were used to collect baseline data. For the post‐initial treatment effectiveness data collection and for the purposes of this study, we adapted the ECLIPSE trial forms by adding an additional WTP question. Both questionnaires were sent to women in the ECLIPSE trial for completion. BODY.METHODS.DATA COLLECTION.ECLIPSE TRIAL BASELINE QUESTIONNAIRE: As part of the trial, follow‐up women were asked to complete the condition‐specific measure MMAS and the generic health‐related quality of life measure EQ‐5D‐3L. The instruments had the following properties: MMAS. MMAS is a self‐report questionnaire that has six attributes including 'practical difficulties', 'social life', 'psychological health', 'physical health and well‐being', 'work/daily routine' and 'family life/relationships'. Each attribute has four levels ranging from unaffected to severely affected. For example, the wording for social life relating to severely affected reads 'My social life is devastated during my cycle. I am unable to make any plans'. The questions refer to 'during my cycle', and the respondent ticks the level that most accurately reflects their experience. The measure is scored on a 0–100 scale, with 0 being worst possible state for the condition and 100 being best possible state. Each attribute has been weighted according to the menorrhagia patients' preferences using 21 counters, which are considered to be importance points. The visual analogue scale (0–100) is then used to weight the relative importance of the levels within the attribute. The weighting for the levels is then multiplied by the weighting for the attribute. The overall score is then derived by summing the value of the levels ticked by the respondent to provide an overall score between 0 and 100.13 EQ‐5D‐3L. EQ‐5D is a generic measure of health outcome that can be used across a range of conditions. Its five attributes include 'mobility', 'self‐care', 'usual activities', 'pain/discomfort' and 'Anxiety/depression' The attributes each have three levels and the questions are asked with reference to 'health today'. Responses to the instrument can be used to generate a health‐related quality of life score referred to as a 'utility' value expressed on a scale where 0 represents death, values below zero worse than death and 1 indicates full health. BODY.METHODS.DATA COLLECTION.POST‐INITIAL TREATMENT EFFECTIVENESS QUESTIONNAIRE: The questionnaire booklets that were designed for the purpose of this study were posted in August 2012 to all eligible women in the ECLIPSE trial. This time point captured women who were either 2 or 5 years post‐initial treatment effectiveness, due to the 3‐year time period for recruitment into the trial. By post, women received (1) a patient information sheet outlining the purpose of the work and; (2) an 'ex‐post' (post‐initial treatment effectiveness) questionnaire; and (3) a prepaid stamped addressed envelope to return the completed questionnaire. The objective of the ex‐post questionnaire was to elicit a WTP value for the pharmaceutical treatment that the women were currently taking, either LNG‐IUS or oral treatment. In this context therefore, average maximum WTP values were derived after the change in outcome had occurred, from respondents who had experience of the condition and experience of the treatment. Hence, WTP was elicited from the ex‐post perspective. The WTP value therefore reflected the direction and level of change in outcome over time in response to treatment. It is a commonly practiced approach to consider use value when eliciting WTP in health care.14, 15, 16 Maximum WTP values were elicited for both LNG‐IUS and oral treatment using the self‐complete ex‐post booklet questionnaire. The booklet was similar in design to the trial questionnaire and captured data on condition‐specific quality of life (MMAS), WTP, socio‐demographic details and health‐related quality of life using the EQ‐5D‐3L. The MMAS was first presented in the questionnaire, followed by questions to determine current and previous treatment taken as part of the ECLIPSE trial. Respondents were asked for their maximum monthly out of pocket WTP value for their current treatment. The time frame of payment of 'up until menopause' was explicitly stated to ensure WTP values were not overestimated.17 To elicit WTP, the payment scale elicitation format was used as it has a higher completion rate than other methods that can be used in a postal questionnaire.18 The scale range used was £0–£500, and an open‐ended option for values greater than £500 was offered. To assess the validity of the WTP responses and the respondents understanding of the WTP question, we then asked the respondent to outline reasons for their WTP values in an open‐ended question. This approach enabled an assessment of responses as well as providing insight into the way in which the WTP question was interpreted. To ensure the WTP values were realistic, that is within the respondent's resources, and in line with good practice, a reminder was included asking the women to consider the amount that they can actually afford to pay.19 BODY.METHODS.ANALYSIS: For the analysis, baseline data were taken from the ECLIPSE trial questionnaire and post‐initial treatment effectiveness data were from the purposely developed WTP questionnaire. Scores were calculated for the baseline MMAS, obtained from the ECLIPSE trial, and the current ('ex‐post') MMAS score. Similarly, baseline and follow‐up EQ‐5D quality of life score was calculated for every woman.20 Descriptive statistics are reported for each measure and a paired t‐test was conducted to determine the difference between follow‐up and baseline values at the 5% level. Cohen's effect size (mean change divided by standard deviation) is calculated for each of the measures where 0.2–0.5 indicates a small change, 0.5–0.8 moderate and >0.8 large.21 As the assessment of the validity of WTP is the aim of this study, and not the incremental difference between treatment arms, the WTP values for both treatments were combined. The CUA and CBA which consider the treatment effect by group are presented elsewhere.12, 22 Thus, the WTP for overall treatment (both LNG‐IUS and oral treatment) for menorrhagia is used to assess the convergent validity. The association between WTP and change in condition‐specific quality of life (MMAS), from baseline to the ex‐post values, was first assessed. Second, the association between WTP and change in general health‐related quality of life as measured by the EQ‐5D was assessed. Finally, the association of change in MMAS and change in EQ‐5D was explored. The associations between the measures were assessed by Spearman's correlation analysis. A Rho value between 0.10 and 0.29 indicates a small association, 0.30–0.49 medium association and greater than 0.5 a large association.23 The percentage improvement in MMAS from baseline to current time point was also calculated, using percentage improvements (<25, 26–50, 51–75 and >75%) to establish the extent to which WTP increased with improvement in MMAS from baseline. The Wilcoxon rank–sum test was carried out to identify whether the differences between the WTP values for each percentage change were significant at the 5% level. The qualitative reasons for the WTP values were further analysed and categories generated based on a previous published WTP study.14 The qualitative information was supplemented by exploring the influence of prominent numbers on the WTP results. Prominent numbers are those that are typically selected by respondents and include £0, 1, 2, 5, 10, 20, 50, 100, 200, 500 and so on.24 The selection of prominent numbers is thought to be related to the respondent's perception of the difficulty of the task and their knowledge of the intervention.25 Where appropriate, GBP is converted to USD using a currency conversion website www.fxexchangerate.com; (£1 = US$0.665; 2013). We carried out all data analyses in STATA (v11.0) and Microsoft Excel. BODY.RESULTS: All of the two hundred and seventy‐two women who were eligible to complete the questionnaire in the trial received a copy. One hundred and sixty‐three questionnaires (60%) were received; however, 78 of these questionnaires were excluded as the women were no longer taking either of the randomized pharmaceutical treatments, and therefore, the WTP section was not applicable to these women. Our intention was to obtain the value for either treatment (LNG‐IUS or oral treatment) and the question posed in the questionnaire referred to 'current treatment'. As these women were either no longer taking any treatment due to menopause or other reasons, it would not be appropriate to use values for 'current treatment'. However, the values of those who have crossed over to the other treatment are included to ensure that the values of those who were unhappy with their original treatment were included. Of the remaining 85 women who returned the questionnaire and were currently taking one of the randomized treatments, 4 (4%) women did not provide a WTP value and MMAS score for their current treatment, and 11 (13%) protest answers were identified from the qualitative explanations offered for the WTP value. Protest responses are defined as an explicit objection to being asked to 'pay' for health care and therefore a misunderstanding of the hypothetical nature of the exercise and are therefore invalid. These 15 non‐responses and protest answers were removed from the analysis. It is generally accepted in the literature that qualitative information associated with the protest responses should be assessed to determine whether the values are genuine WTP values or whether the respondent is protesting against the exercise. There is a debate in the literature about the inclusion of protest responses.14 Where protests are identified, it has been argued that these should not be included in the analysis as they are not valid responses; however, some authors have expressed concerns about their exclusion if the characteristics of the excluded and included group are not assessed.14 There were no significant differences between the characteristics of this excluded group to the sample analysed (see online supplementary material). In total, 70 respondents gave a WTP value for either LNG‐IUS or oral treatment and the characteristics of this sample are presented in Table 1. The proportion of respondents that had a household income of less than £30 000 (US$45 113) was approximately 50%, which was lower than the national average where 65% are below approximately £27 000 (US$40 602).26 Table 1 Sample characteristics of WTP respondents Variable Treatment ( n = 70) Expected age of menopause (yrs) [SD] 53.80 [2.50] Age [SD] 48.09 [3.93] Marital status (%) Married or living with partner 53 (76) Not 17 (24) Employment status (%) Employed (FT)/(PT) 56 (80) Not 14 (20) Household income (%) Less than £20 000 (<$30 075) 22 (32) £20 001–30 000 ($30 077–$45 113) 14 (20) £30 001–40 000 ($45 114–$60 150) 10 (14) £40 001–50 000 ($60 152–$75 188) 9 (13) More than £50 000 (>$75 189) 13 (20) Main earner (%) Yes 32 (46) No 37 (54) FT, full‐time; PT, part‐time; SD, standard deviation. (US$). John Wiley & Sons, Ltd BODY.RESULTS.TREATMENT RESPONSE: The average maximum WTP for treatment for menorrhagia was approximately £27 (US$41) per month (see Table 2). The average combined MMAS score for treatment doubled from approximately 43 at pre‐treatment to 85 following treatment, generating a statistically significant improvement in health status as measured by this instrument (P = 0.000). According to the Cohen's standardized effect size, the mean effect of 1.97 observed on MMAS would indicate a large change as the effect size is greater than 0.8. Whilst average EQ‐5D values increased significantly (P = 0.0168) from 0.789 pre‐treatment to 0.880 post‐initial treatment effectiveness, the mean effect size of 0.36 indicates a small change using the same Cohen's criteria (Table 3). Whilst 0.789 pre‐treatment to 0.880 post‐initial treatment effectiveness generates a mean effect size of 0.36 which would indicate a small change using the same Cohen's criteria, it should be noted that this level of change in EQ‐5D would lead to a change in one level of one attribute; for example, using the pain attribute a change would be observed from some pain to no pain which could be deemed clinically important. Table 2 Descriptive statistics for WTP Valid responses Mean (Median) [$] Min–Max [$] SD [$] WTP 70 £26.99 (£10) [$40.59 ($15)] £0–£500 [$0–$752] £60.73 [$91.32] SD, standard deviation; WTP, willingness‐to‐pay. (US$). John Wiley & Sons, Ltd Table 3 Average scores for the instruments Mean MMAS [SD] EQ‐5D [SD] WTP ($) Baseline ( n = 70) 43.23[21.22] 0.789 [0.250] Post‐initial treatment effectiveness ( n = 70) 85.00 [23.15] 0.880 [0.215] £26.99 ($40.59) Change ( n = 70) 41.77 [32.03] 0.09 [0.31] MMAS, Menorrhagia multi‐attribute scale; SD, standard deviation; WTP, willingness‐to‐pay. John Wiley & Sons, Ltd BODY.RESULTS.RESPONDENT'S UNDERSTANDING OF WTP: Among the 70 women who provided a WTP value, 69 provided a reason for the value. Nine categories of reasons for a WTP value were generated from the qualitative information from all of the women, which included protests and non‐response. The categories of reasons for the sample analysed are presented in Table 4. It can be seen that for both LNG‐IUS and oral treatment that 'R4: affordability' and 'R7: effects of treatment' are the most commonly cited reason for a WTP value. In addition to this finding, it can be seen from Fig. 1, which presents the WTP values for treatment against the number of observations, that the most commonly selected WTP values were prominent numbers, those are £10 and £20, which make up 31 and 16% of the sample which could indicate difficulty with the WTP exercise or a tendency to round numbers (discussed later). Table 4 Explanation given for WTP value (sample analysed, ex‐post) Category Explanation Total n (%) R2 Subject expressed difficulty estimating WTP owing to: Difficult to answer Cannot put a price on health care 8 (7) R3 WTP based on nominal amount Arbitrary sum/guess/out of thin air 1 (0.9) R4 WTP reflects ability to pay (affordability) Maximum affordable amount given current situation 37 (35) R5 WTP reflects reasonable value NHS should pay but this is a reasonable limit 8 (7) R6 WTP reflects cost of treatment Attempted to estimate cost Used a comparator such as prescription costs 5 (5) R7 WTP reflects effect of treatment In terms of effectiveness outcomes In terms of process utility 30 (28) R9 Related to cost of sanitary wear Washing clothes/wipes/painkillers 13 (12) R10 Misunderstood exercise but provided WTP value 5 (5) Total 107 a NHS; National Health Service, WTP; willingness‐to‐pay. a Some respondents gave more than one reason for their WTP value. Categories R1 and R8 were related to protest responses and are deleted from the table as they were not included in the sample analysed. John Wiley & Sons, Ltd Figure 1Frequency of WTP values. BODY.RESULTS.ASSOCIATIONS: Average maximum WTP was significantly positively correlated with change in MMAS (P = 0.025) (Table 5). That is, the greater the change in health state, as measured by change in MMAS, the greater the WTP value. However, the strength of the relationship is relatively small, generating a rho value of 0.27. In contrast, the association between change in EQ‐5D and change in MMAS did not show statistical significance (P = 0.059), despite demonstrating a positive correlation (r = 0.23). An unusual, though non‐significant result is generated in the association between WTP and change in EQ‐5D, as a minor negative correlation (r = −0.02) is observed. Table 5 Associations between measures Change in MMAS (rho) WTP (rho) Change in EQ‐5D (rho) Change in MMAS 1.0000 WTP 0.2674 a 1.000 Change in EQ‐5D 0.2265 −0.0158 1.000 a P = 0.0252 (<0.05) MMAS; menorrhagia multi‐attribute scale, WTP; willingness‐to‐pay. John Wiley & Sons, Ltd When WTP for percentage change in MMAS is assessed, the mean WTP increases as the change in health status increases (from approximately £16 to £63 (US$25–US$95)). Whilst the mean values would suggest that WTP would continue to increase the greater the change in health status, the median values illustrate the skewness of the data but still demonstrate an increase in WTP as health status improves (Table 6). The significance tests show the WTP values between '<25%' and '51–75%' to be significantly increased as percentage change in MMAS increases (P = 0.033; P < 0.05). Table 6 Mean WTP against percentage improvement in MMAS % change in MMAS Mean MMAS [SD] No. obs Mean WTP [SD] ($) Median ($) Min–Max WTP ($) <25 1.27 [12.82] 21 £16.29 [£17.97] ($24.50) £10 ($15) £0–£50 ($0–75) 26–50 36.13 [8.3] 14 £20.86 [£24.65] ($31.37) £10 ($15) £6–£100 ($9–$150) 51–75 60.95 [9.04] 24 £23.38 [£20.63] ($35.16) £20 ($30) £8–£100 ($5–$150) >75 84.44 [10.00] 11 £63.09 [£145.86] ($94.87) £20 ($30) £0–£500 ($0–$752) MMAS; menorrhagia multi‐attribute scale, SD, standard deviation; WTP; willingness‐to‐pay. (US$). John Wiley & Sons, Ltd BODY.DISCUSSION: In this exploratory study, we aimed to assess the convergent validity of WTP within the context of menorrhagia by comparing change in the condition‐specific measure (MMAS) to (1) change in WTP and (2) the generic health‐related quality of life measure, EQ‐5D‐3L. Overall, our findings suggest the convergent validity of WTP as it behaves as would intuitvely be expected in response to change in health status, as measured by MMAS. Specifically, following treatment, WTP increases with a greater improvement in treatment benefit, and the correlation between the change in condition‐specific MMAS and WTP suggests statistical significance. An association between MMAS and the generic measure, EQ‐5D, was not observed as the change in EQ‐5D scores before and after treatment was not significantly associated with the equivalent change in MMAS. This result suggests that WTP is more sensitive to change in the condition‐specific measure (MMAS), than the generic measure (EQ‐5D). We hypothesize that this finding is attributed to the EQ‐5D instrument being designed as a generic health‐related quality of life measure, which is not focussed specifically on menorrhagia. We suggest that WTP is more sensitive given that it has the potential to measure both health and non‐health aspects of quality of life that are important to women who suffer with menorrhagia, which are also encompassed by the MMAS measure. Qualitative reasons for the WTP responses provided a further opportunity to assess the reliability of the WTP values. The analyses of the reasons confirmed that respondents were considering the 'value' of the treatment as was theoretically expected. These values are therefore reflective of what the theory says people consider when completing WTP exercises. However, the selection of prominent numbers could be related to the perceived difficulty of the task 25 where respondents provide less precise values when they do not feel they have adequate knowledge of the good being valued.24 In this study, the WTP values were elicited from the ex‐post perspective where respondents have experience of, and are knowledgeable about, the treatments and despite this, prominent numbers were most commonly selected. This indicates that although respondents stated 'true' WTP values as confirmed by the qualitative responses, the selection of prominent numbers alludes to the WTP elicitation task being difficult to complete. Finally, as there are several possible approaches for eliciting WTP, the method used in the analysis reported here requires some justification. With respect to the WTP question, the time period 'up until menopause' seemed intuitive given that menorrhagia ceases at menopause and the scale of £0–£500 was thought most suitable, given that the questionnaire asked respondents to provide a monthly WTP value. The monthly payment time frame was used because women generally pay monthly (or every 3 months) for prescriptions for menorrhagia, for sanitary protection and they experience the benefits of treatment on a monthly basis. The out of pocket payment vehicle was deemed appropriate for this context because whilst the full price for treatment is not paid in the UK, patients do make an out of pocket payment for prescriptions for oral treatment in the UK. Although this private payment does not exist for LNG‐IUS, the existence of private payment within this context is likely to minimize the issue of hypothetical bias. To our knowledge, this is the first study to assess the convergent validity of WTP against a change in condition‐specific quality of life measure in menorrhagia. It is also the first to compare the correlation of WTP and change in EQ‐5D with a condition‐specific quality of life measure. A limitation of this exploratory study is the sample size used. Given that the significant difference was observed between the two percentage change categories with the greatest number of observations, it is likely that the remaining categories were not found to be significant due to the limited sample size for the groups '26–50%' and '>75%'. Thus, there may not have been sufficient power to detect a significant difference between these groups. The findings from this exploratory study, however, can be used to inform future sample size calculations for subsequent WTP studies in the area. Prior to this study, it was not readily possible to calculate sample size requirements as a priori data on the distribution of WTP values for the LNG‐IUS or oral treatment were not available.27 This study therefore enables researchers to identify the number and range of responses given to determine how many respondents are required to detect a certain difference in WTP across treatments, that is the WTP value that constitutes a meaningful difference in improvements from baseline or between treatments. To our knowledge, no other study has assessed the sensitivity to scale of WTP according to a longitudinal change in outcome measured by a condition‐specific measure. Other related studies have, however, assessed the relative sensitivity of WTP and time trade‐off (TTO) for changes in described dimensions of health states and have tentatively suggested that WTP is sensitive to change in different levels of health within the same dimension.9 The sensitivity of (ex ante) WTP with a condition‐specific measure at one time point has also been explored.28, 29 Radtke et al.28 assessed the relationship between WTP, EQ‐5D and a condition‐specific measure for vitiligo; and Schiffner et al.29 assessed the sensitivity of WTP and TTO with a condition‐specific measure for psoriasis. Baseline EQ‐5D (or TTO) and condition‐specific scores for current health state were identified, and hypothetical WTP values were elicited for a cure. Both studies found that WTP had a significant correlation with the condition‐specific measure, and Radtke et al.28 also showed WTP and EQ‐5D to have negative correlation. Other psychometric properties of WTP and EQ‐5D in menorrhagia have also been explored. A review of psychometric properties of measures used in menorrhagia3 identified four key studies. One study assessed the consistency of responses of WTP from women with menorrhagia and found a lack of external reliability of WTP.30 The second found EQ‐5D to be unsuitable for patients with menorrhagia.4 In the third, MMAS was statistically associated with changes in satisfaction post‐initial treatment effectiveness, whilst EQ‐5D was not.31 Finally, a lack of sensitivity of EQ‐5D to changes in quality of life associated with menorrhagia was observed.32 Further research is required to consolidate our findings using a larger sample size, the requirements for which can now be derived based on our study. Additional research could explore the convergent validity of other methods of valuation such as SF‐6D in such a condition, assessing changes in SF‐6D in relation to WTP and MMAS to determine the extent to which SF‐6D reflects changes in MMAS. This exploratory study suggests that there is the potential, and a benefit, to the use of WTP in chronic conditions with episodic symptoms which impact on health and non‐health aspects of life. BODY.CONFLICTS OF INTEREST: The authors declare that they have no conflict of interest. BODY.SOURCE OF FUNDING: This study research relates to a PhD studentship which was funded by the National Institute for Health Research Health Technology Assessment programme, UK. BODY.SUPPORTING INFORMATION: Table S1. Characteristics of sample analysed and excluded respondentsClick here for additional data file.

TITLE: Effects of a balance-based exergaming intervention using the Kinect sensor on posture stability in individuals with Parkinson’s disease: a single-blinded randomized controlled trial ABSTRACT.BACKGROUND: The present study examined the effects of a balance-based exergaming intervention using the Kinect sensor on postural stability and balance in people with Parkinson's disease (PD). ABSTRACT.METHODS: We conducted a subject-blinded, randomized controlled study. Twenty people with PD (Hoehn and Yahr stages I through III) were recruited and randomly assigned to either a balance-based exergaming group (N = 10) or a balance training group (N = 10) for an 8-week balance training period. Postural stability was assessed using the limits of stability (LOS) and one-leg stance (OLS) tests. Balance was assessed using the Berg Balance Scale (BBS) and the timed up and go (TUG) test. Participants were assessed pre- and post-training. ABSTRACT.RESULTS: After training, participants in the balance-based exergaming group showed significant improvements in LOS performance, and in the eyes-closed condition of the OLS test. Both training programs led to improvements in BBS and TUG performance. Furthermore, balance-based exergaming training resulted in significantly better performance in directional control in the LOS test (78.9 ± 7.65 %) compared with conventional balance training (70.6 ± 9.37 %). ABSTRACT.CONCLUSIONS: Balance-based exergaming training resulted in a greater improvement in postural stability compared with conventional balance training. Our results support the therapeutic use of exergaming aided by the Kinect sensor in people with PD. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov.NCT02671396 ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12984-016-0185-y) contains supplementary material, which is available to authorized users. BODY.BACKGROUND: People with idiopathic Parkinson's disease (PD) commonly exhibit postural instability during daily activities [1]. PD-related balance impairment is associated with a loss of mobility and increased likelihood of falls, and can cause marked disability [2, 3]. To ameliorate postural instability, techniques using external feedback with cueing or sensory stimuli have been investigated [4, 5]. Several studies suggest that external feedback may initiate other neural pathways and play a significant role in the volitional control of movements for people with PD [6, 7]. Virtual reality (VR) technologies such as exergaming may have therapeutic value in the treatment of postural instability [8–10]. VR is a technology that allows the user to interact directly with a computer-simulated environment [11]. Exergames are computer games that are controlled by body movements. VR and exergaming can provide augmented feedback in real time, while a person performs specific motor tasks [12]. Opportunities for repeated accurate performance can be incorporated into VR and exergaming to enhance motor learning [7, 13]. Moreover, VR games can be effective for retaining participants' interest and motivation. A recent meta-analysis suggested that exergaming may provide an appropriate training approach to improve balance and functional mobility in healthy older people [14]. These findings raise the possibility that exergaming might also provide an approach for improving postural instability for people with PD. A previous study examined the effects a 6-week home-based balance training program using the Wii Fit game for a total of 18 training sessions on balance and functional abilities in people with PD, compared with a group of paired healthy participants [15]. Another study investigated the effects of Wii-based training compared with conventional balance training for 7 weeks (a total of 14 training sessions) on activities of daily living in people with PD [16]. Both studies revealed positive effects of exergaming on balance, functional abilities and activities of daily living among people with PD. However, positive effects were found only within groups, with no between-group differences observed in a comparison with the control group. The absence of between-group differences may have resulted from an inability to capture the full-body motion involved in postural control, or the lack of a sufficiently sensitive sensor to accurately measure motion. The shortcomings of the Wii system's sensors may limit its potential as an effective intervention [17]. A new exergaming system was recently developed using the Kinect sensor. The Kinect sensor is a low-cost device that can provide measurements for most of the main human joints. Previous studies reported that a kinematic measurement method using the Kinect sensor was accurate and reliable for measuring postural control [18, 19]. These findings suggest that the Kinect sensor could provide a useful tool for therapeutic use. However, there has been little research into the therapeutic use of the Kinect sensor to date. The present study sought to test a therapeutic application of exergaming using the Kinect sensor. We examined the effects of an 8-week balance-based exergaming program developed in our lab, compared with an 8-week period of conventional balance training (16 training sessions), on postural stability and balance in people with PD. We hypothesized that participants who underwent an 8-week balance-based exergaming intervention would demonstrate superior performance on measures of postural stability and balance, compared with those who received balance training. BODY.METHODS.PARTICIPANTS: Participants were recruited from Mackay Memorial Hospital in Taipei. Outpatients with PD were informed about the study by a neurologist. Eligibility required a diagnosis of idiopathic PD according to the United Kingdom Brain Bank Criteria [20] by the same neurologist. Information on age, gender, the more affected side, and disease duration were obtained through patient interviews and from medical charts. All participants met the following inclusion criteria: (1) Hoehn and Yahr stages I through III, (2) a score of ≥ 24 on the mini-mental state examination, (3) stable medication usage and (4) standing unaided to perform the measurement and training. The exclusion criteria were as follows: (1) histories of other neurological, cardiovascular, or orthopedic diseases affecting postural stability and (2) uncontrolled chronic diseases. In total, 48 individuals were identified as potential participants for this study. Of these, 22 participants gave informed consent and participated in the study. BODY.METHODS.STUDY DESIGN: This study was a subject-blinded, randomized controlled trial. The study protocol was approved by the Institutional Review Board of Mackay Memorial Hospital (reference number: 13MMHIS120) and was explained to all participants before their participation. The study was performed in accordance with the Declaration of Helsinki. Block randomization was used to assign participants to either the balance-based exergaming (BE) or the conventional balance training (BT) group. Assignment was performed by an independent person who selected one of a set of sealed envelopes 30 min before the intervention began. Participants in the BE and BT groups received an 8-week balance-based exergaming intervention, and conventional balance training, respectively. Measures of postural stability and functional balance were measured pre- and post-training. The measurement and intervention were conducted with participants in the "on" state, when they were moving freely and easily without dystonia, excessive rigidity or tremor. The data were collected in a university laboratory. BODY.METHODS.INTERVENTION: Participants in both groups underwent balance training for 50 min per session, two sessions every week, for 8 weeks. Each training session began with a 10-min warm-up and ended with a 10-min cool-down. Both the warm-up and cool-down periods focused on stretching exercises of the trunk and extremities. Participants in the BE group received a 30-min balance-based exergaming intervention using the Kinect sensor (Microsoft Corporation, Redmond, WA, USA). The Kinect sensor incorporates infrared light and a video camera, which creates a 3D map of the area in front of it. This device provides full-body 3D motion capture. Four exergaming programs were used for training (Fig. 1), designed to incorporate an appropriate level of challenge to match the ability and fitness of people with PD. The first program was called Reaching task 1. In this task, participants were asked to reach toward a stationary target at a given location. The second program was called Reaching task 2. Participants were asked to track a moving object by lengthening the arm and immersing the hand into the object as it flew in 3D space. The third program was called Obstacle avoidance. Participants were instructed to avoid upcoming obstacles that approached from varying directions at random, by moving the body right/left or up/down. The final task was called Marching. Participants were instructed to step alternately without going forward or backward while following dynamic bars that were automatically rising and falling at a predetermined speed and frequency. During the training duration, the challenge level was increased progressively by adjusting the amplitude, frequency, speed, complexity and number of hints. The details of the exergaming programs are shown in Table 1.Fig. 1Screen shots of interaction with the exergaming program. Four exergaming programs, Reaching task 1 (a), Reaching task 2 (b), Obstacle avoidance (c) and Marching (d), were designed and used for trainingTable 1Program of balance-based exergaming interventionProgramActionProgressionMotor demandReaching task 1Standing in a given area and reaching toward a stationary target at different heights, depths and in different directions• Reaching length• Number of targets• Range of distribution• Amount of repetition• Weight shifting• Challenging limits of stability• Functional transitionsReaching task 2Standing in a given area and tracking a moving object while extending arm and immersing the hand into the object as it flew in 3D space• Speed• Moving range• Pathway pattern• Remembered sequence or course of trajectory• Weight shifting• Arm coordination• Advance motor planningObstacle avoidanceStanding in a given area and preparing to avoid upcoming obstacles that randomly approached from varying directions by moving body sideways or up/down• Obstacle hitting ratio• Speed• Dual task• Hitting direction• Quick change strategy• Movement adaption• AgilityMarchingAlternating steps without going forward while following dynamic bars that automatically rose and fell at a predetermined speed and frequency• Frequency• Gap between steps• Functional stepping• Leg coordination• Single limb support Participants in the BT group underwent a 30-min conventional balance training session. The training program included reaching activities, weight-shifting activities and marching activities. The general training protocols used for the BT group were the same as those used for the BE group. The challenge level was increased progressively by changing the base of support, speed, complexity and deprivation of sensory inputs. BODY.METHODS.OUTCOME MEASURES.POSTURAL STABILITY: The limits of stability (LOS) and one-leg stance (OLS) tests were used to assess postural stability in this study. Participants were harnessed into a suspension system to prevent falls when performing the tasks. LOS performance was measured using the Smart Balance Master (NeuroCom International Inc., Clackamas, OR, USA) instrument to extract quantitative data [21–24]. The LOS test provides an assessment of the ability to intentionally displace the center of gravity (COG) to the participant's stability limits without losing balance. In this task, participants were asked to quickly transfer their COG, while standing on stable force plates, toward eight targets spaced at 45° intervals around the COG, represented on a computer monitor. All participants underwent one practice trial followed by one test trial. In the LOS test, we measured reaction time (the time from the presentation of a start cue to the onset of the voluntary shifting of the participant's COG toward the target position), movement velocity (average speed of COG movement based on the middle 90 % of the distance, measured in degrees per second), end point excursion (percentage of the distance achieved toward a target on the initial movement) and directional control (100 % being a straight line from the center of pressure to the intended target). The validity and reliability of the LOS test in people with neurological disease has been well established [25–27]. The OLS test is an assessment of postural steadiness [15, 28–31]. Participants were asked to cross their arms over the chest, and to stand on either the less or more affected leg, with the other leg raised so that the raised foot was near but not touching the ankle of the stance leg. The assessor timed the OLS test until participants either: (1) uncrossed the arms, (2) moved the stance leg, (3) moved the raised leg touching the floor or the stance leg, (4) opened the eyes on eyes-closed trials or (5) reached a maximum of 30 s. Each participant performed three trials with the eyes open, and three trials with the eyes closed. Data were averaged from the three trials. A previous study found a high degree of reliability (ICC = 0.87) in the OLS test in older adults [32]. BODY.METHODS.OUTCOME MEASURES.FUNCTIONAL BALANCE: The Berg Balance Scale (BBS) and the timed up and go (TUG) test were used to assess functional balance. The BBS comprises a set of 14 balance-related tasks, ranging from standing up from a sitting position, to standing on one foot. The degree of success in each task is given a score from zero (unable) to four (independent), and the final measure is the sum of all scores. The highest possible score on the BBS is 56, which indicates excellent balance. The validity and reliability (ICC > 0.95) of BBS scores in people with PD has been established in several studies [33–35]. The TUG test is a mobility test requiring both static and dynamic balance. During the test, the assessors measured the time participants took to rise from a chair, walk 3 meters, turn around, walk back to the chair, and sit down. Each participant performed three trials of the TUG test. Data were averaged from the three trials. The TUG test has previously been found to have high validity and reliability (ICC > 0.87) for assessing balance in people with PD [36, 37]. BODY.METHODS.SAMPLE SIZE: The sample size calculation was based on a pilot study that tested eight participants at Hoehn and Yahr stages 1 and 2, indicating a difference of 0.2 s between pre- and post-training on reaction time in the LOS test. Based on this difference, a sample size calculation indicated that 20 participants would be sufficient for 85 % power (α = 0.05). BODY.METHODS.STATISTICAL ANALYSIS: All analyses were performed using the SPSS 20.0 statistical package (SPSS Inc., Chicago, IL, USA). Descriptive statistics were generated for all variables, and distributions of variables were expressed as the mean ± standard deviation. Because of the relatively small number of participants included in the current study (N < 30) and since the results of a Shapiro-Wilk test did not allow us to assume that the data were normally distributed, nonparametric tests were employed. Comparison of two groups for general characteristics was made using chi-square or Mann-Whitney U test for categorical or continuous variables, respectively. The Friedman test, followed by a post hoc test, was used to determine differences in each dependent variable. The Wilcoxon signed-rank post hoc test was performed for within-group comparisons and the Mann-Whitney U post hoc test was performed for between-group comparisons. The statistical significance was set at P ≤ 0.05. BODY.RESULTS: A total of 48 individuals were screened and 22 enrolled between 2013 and 2014. Of these, 11 were assigned to the BT group, and 11 were assigned to the BE group. Of 22 participants, two did not complete the intervention (one in the BT group and one in the BE group). A flow diagram of the study protocol is shown in Fig. 2. The 20 participants who completed the intervention attended all intervention sessions. None of the participants reported any adverse events.Fig. 2Flowchart of the experimental design The demographic characteristics of participants in both groups are presented in Table 2. Demographic differences between the two groups were not significant. Moreover, differences in all pre-intervention-selected outcome measures in the two groups were not significant (Table 3).Table 2Baseline demographics and clinical characteristics of the subjectsBalance-based exergaming group (N = 10)Balance training group (N = 10) P Age (years)67.5 ± 9.9668.8 ± 9.670.67Sex (male/female)9/17/30.58Disease duration (years)4.03 ± 3.745.22 ± 4.850.34Hoehn and Yahr stage1.6 ± 0.841.4 ± 0.520.73Mini-Mental State Examination27.4 ± 2.5928.2 ± 1.990.40More affected side (right/left)8/25/50.35Data are presented as the mean ± standard deviation or proportionTable 3Outcome measures for each groupBalance-based exergaming group (N = 10)Balance training group (N = 10)Friedman testPre-trainingPost-trainingPre-trainingPost-training P Limits of stability Reaction time (sec)0.96 ± 0.330.74 ± 0.24*0.88 ± 0.240.79 ± 0.18<0.001 Movement velocity (deg/sec)3.37 ± 1.353.83 ± 0.974.19 ± 1.544.57 ± 1.410.07 Endpoint excursion (%)75.2 ± 12.4884 ± 12.04*79.7 ± 13.8481.8 ± 11.370.04 Directional control (%)75.7 ± 8.7878.9 ± 7.65*,† 70.9 ± 10.8570.6 ± 9.370.02One-leg stance Less affected with eyes open (sec)17.39 ± 12.8715.16 ± 10.539.14 ± 9.6312.98 ± 11.080.47 More affected with eyes open (sec)15.06 ± 11.2315.58 ± 11.5813.72 ± 12.4314.54 ± 9.650.09 Less affected with eyes closed (sec)3.35 ± 2.856.1 ± 8.65*2.71 ± 2.545.31 ± 7.680.002 More affected with eyes closed (sec)3.06 ± 2.554.13 ± 2.745.88 ± 7.566.66 ± 8.410.16Berg Balance Scale50.9 ± 5.3253.2 ± 2.86*50.4 ± 4.7953 ± 1.89*0.001Timed up and go (sec)9.5 ± 2.458.71 ± 1.8*10.05 ± 4.669.18 ± 3.42*0.007Data are presented as mean ± standard deviation*and †are P ≤ 0.05 for within-group and between-group comparisons, respectively The results of the interventions are presented in Table 3. Analysis of selected outcomes using the Friedman test revealed a significant effect of intervention type on reaction time, endpoint excursion and directional control in the LOS test, and in the less affected leg in the eyes-closed condition in the OLS test, the BBS and the TUG test. Within-group post hoc analysis revealed that balance-based exergaming training significantly improved LOS performance (improving reaction time from 0.96 ± 0.33 to 0.74 ± 0.24 s, end point excursion from 75.2 ± 12.48 to 84 ± 12.04 % and directional control from 75.7 ± 8.78 to 78.9 ± 7.65 %) and OLS on the less affected leg in the eyes-closed condition (from 3.35 ± 2.85 to 6.1 ± 8.65 s). Compared with the BT group (70.6 ± 9.37 %), the BE group (78.9 ± 7.65 %) exhibited better performance in directional control of LOS post-training. Functional balance in both groups, as measured by the BBS and the TUG test, was improved significantly post-training compared with pre-training. However, no significant differences were found between groups. BODY.DISCUSSION: This study produced two main findings: (1) balance-based exergaming training had a greater effect on postural stability compared with conventional balance training; and (2) both training programs improved functional balance in people with PD. The current study tested two balance training programs with similar training protocols. A recent meta-analysis examined the BBS, postural sway, TUG, and Functional Reach test as measures of postural stability, reporting that exercise therapy is an important treatment option for improving postural stability in people with PD [38]. The findings suggested that exercises containing a balance component were most beneficial in improving postural stability in people with PD [38]. In the current study, we used the LOS and OLS tests to measure postural stability, and the BBS and TUG tests to measure functional balance. The current findings were in line with the findings of Klamroth et al., who reported that balance training was beneficial for performance in the BBS and TUG tests [38]. Our findings revealed that only balance-based exergaming training produced positive effects on LOS and OLS, with particularly strong effects on directional control in LOS. These findings suggest that exergaming training using the Kinect sensor contributed to the beneficial gains we observed. As a therapeutic tool, the Kinect sensor can provide specific motor practice using full-body motion capture, which offers precise real-time information to guide performance and monitor body movement. Previous clinical trials indicated that exergaming programs using the Kinect sensor resulted in accurate capture of movement components [39, 40]. Our results revealed within-group improvements on most measures of postural stability during the exergaming intervention training period. Our exergaming programs involved various balance challenges. This may have contributed to our positive findings, involving actions focused on agility, challenging postural or locomotor-like skills, and reaching away from the base of support. All of these are involved in whole-body movements. In addition, the repetitive, real-time feedback and graded complexity in our exergaming programs may have contributed to the positive effects of training reflected in LOS performance. However, the movement velocity of LOS remained unchanged after exergaming training. Persistent bradykinesia [41] and a choice to focus on improving accuracy rather than faster motor performance among people with PD are possible reasons for our movement velocity findings [21]. The current results also revealed better OLS performance in the eyes-closed condition after exergaming training. A previous study using a Wii-based system reported similar results [15]. Because participants needed to focus on each joint position while carrying out the fine motor plan necessary for many of the tasks in the exergaming training, stimulation of proprioceptive feedback or an improvement in the internal representation of balance may have enhanced OLS performance. Little evidence is available regarding the minimal clinically important differences in postural stability and balance outcomes in people with PD. Evidence of minimal clinically important differences for LOS and OLS test in PD is lacking. Steffen and Seney reported a minimal detectable change of 5 points on the BBS for people with PD [34]. In the current study, we recorded a 2.45-point improvement after balance training for BBS. The minimally detectable change in TUG performance in people with PD has previously been reported to be 3.5 s [42], which is greater than the 0.83-second improvement observed in the present study. The small but significant changes observed in this study support the therapeutic use of exergaming interventions. However, a greater evidence base is required to support the clinical significance of these results. Several important characteristics have been identified for useful interventions in PD, suggesting that interventions should be task-specific, progressive, variable in terms of practice, and highly challenging [43, 44]. The exergaming programs designed for the current study involved each of these components. For specificity, the full-body motion capture method can be tailored for the needs of balance strategies. To create an appropriate practice resource and construct the progression and variability of program, we implemented enriched setting parameters by increasing speed, repetition and the addition of tasks. Additionally, the novel motor training gave participants more experience and an opportunity to explore or learn to negotiate the new challenges. Although only directional control in the LOS test showed a significant between-group difference, exergaming training using the Kinect system may provide additional benefits. Participants are able to practice free motions without wearing a sensor that could cause discomfort and inconvenience. Reduced staff intervention and the affordability of the device are important economic benefits of the system. Finally, considering the clinical implications of our findings, the current results suggest that the Kinect system can provide an assistive modality with therapeutic potential as a training tool under the supervision of a therapist. The current study involved several limitations. First, the sample size was small, limiting the strength to interpret our results. Second, calibration variability was observed during the preparation of each exergaming session. This issue may have influenced the effect of training because calibration was used to normalize each participant's body information. This formed the basis of the exergaming programs that were tailored for individuals with varying levels of ability. Third, most participants in this study exhibited only mild impairment, and performance at baseline was relatively high. This may have limited the benefits received from training, and the generalizability of our findings to the target population. Finally, the absence of kinematic data meant we were unable to examine spatio-temporal changes in detailed movements. BODY.CONCLUSION: The current study revealed that an 8-week period of balance-based exergaming training using the Kinect sensor resulted in a greater improvement of postural stability than conventional balance training. Both exergaming and conventional balance training had positive effects on functional balance. This trial supports the potential therapeutic use of exergaming aided by the Kinect sensor for people with PD. Importantly, the significant changes in BBS and TUG performance observed after both the exergaming and conventional balance training did not reach the minimal detectable change in patients with PD. Further studies on the use of exergaming are needed to verify the clinical implications of these results.

TITLE: Social Markers of Mild Cognitive Impairment: Proportion of Word Counts in Free Conversational Speech ABSTRACT: Background: Detecting early signs of Alzheimer's disease (AD) and mild cognitive impairment (MCI) during the pre-symptomatic phase is becoming increasingly important for cost-effective clinical trials and also for deriving maximum benefit from currently available treatment strategies. However, distinguishing early signs of MCI from normal cognitive aging is difficult. Biomarkers have been extensively examined as early indicators of the pathological process for AD, but assessing these biomarkers is expensive and challenging to apply widely among pre-symptomatic community dwelling older adults. Here we propose assessment of social markers, which could provide an alternative or complementary and ecologically valid strategy for identifying the pre-symptomatic phase leading to MCI and AD. Methods: The data came from a larger randomized controlled clinical trial (RCT), where we examined whether daily conversational interactions using remote video telecommunications software could improve cognitive functions of older adult participants. We assessed the proportion of words generated by participants out of total words produced by both participants and staff interviewers using transcribed conversations during the intervention trial as an indicator of how two people (participants and interviewers) interact with each other in one-on-one conversations. We examined whether the proportion differed between those with intact cognition and MCI, using first, generalized estimating equations with the proportion as outcome, and second, logistic regression models with cognitive status as outcome in order to estimate the area under ROC curve (ROC AUC). Results: Compared to those with normal cognitive function, MCI participants generated a greater proportion of words out of the total number of words during the timed conversation sessions (p=0.01). This difference remained after controlling for participant age, gender, interviewer and time of assessment (p=0.03). The logistic regression models showed the ROC AUC of identifying MCI (vs. normals) was 0.71 (95% Confidence Interval: 0.54 – 0.89) when average proportion of word counts spoken by subjects was included univariately into the model. Conclusion: An ecologically valid social marker such as the proportion of spoken words produced during spontaneous conversations may be sensitive to transitions from normal cognition to MCI. BODY.INTRODUCTION: High value is given to detecting early signs indicating the transition from normal cognitive aging to Mild Cognitive Impairment (MCI) when early intervention and treatment against Alzheimer's disease (AD) could be most effective. However, it is difficult to distinguish early signs of MCI from normal cognitive aging. Although biomarkers such as CSF beta-amyloid, tau and neuroimaging markers have been extensively examined as early indicators of the pathological process for AD, assessing these biomarkers is expensive and challenging to apply widely among pre-symptomatic older adults. Social behavioral markers (which we propose to call "social markers", a subset of behavioral biomarkers) offer a cost-effective alternative or complementary tool for detection of the transition from normal cognition to MCI in community dwelling older adults. One area of research that has been receiving a lot of attention is assessment of speech characteristics. Recent rapid technological advancement in the area of social interactions research could potentially serve to facilitate the identification of easily measured aspects of conversation that could reflect early cognitive changes in at risk older adults. This includes social network analysis at the macro level (e.g., who contacts whom and how this network pattern or structural network size changes as individuals transit from normal cognition to MCI) and analysis of spoken language at the micro level [1-4]. The present study is a part of a larger randomized controlled clinical trial (RCT) that assessed whether frequent conversations via webcam and Internet-enabled personal computers could improve cognitive function in older persons with either normal cognition or MCI (ClinicalTirals.gov registration number: NCT01571427). The study protocol and the results has been described in detail elsewhere [5, 6]. Briefly, in the larger intervention trial, social interaction sessions were conducted using semi-structured conversations with trained interviewers for 30~35 minutes a day, 5 days a week for 6 weeks (i.e., 30 sessions) among the intervention group. The intervention group also completed a weekly Internet survey that assessed social engagement activities during the previous week, while the control group was contacted by phone to complete the same weekly survey. This weekly telephone call was the only contact the control group had through the trial. Our primary outcome was to assess changes in cognitive functions measured by neuropsychological tests and our secondary outcome was to assess changes in psychological well-being. The trial showed that adherence to the protocol among the intervention group was high (89%; range, 77%-100%). At the post-trial assessment, the normal cognition group assigned to the intervention improved on tests of language-based executive function (e.g., verbal fluency category) compared to normal controls [5]. During the trial, it came to our attention that interviewers had more difficulty changing conversation topics or ending conversation sessions with some participants. Given that one of our aims of the trial was to standardize the interviews across interviewers, we decided to further investigate the observed variability in conversational flow in this follow-up study. Our hypothesis was that MCI individuals would have an impaired ability to identify social cues required for smooth interactions and would have difficulty taking turns in conversation (e.g., keep talking until being interrupted by interviewers). Previous research indicates that executive functioning underlies many everyday activities that are difficult for individuals with MCI [7]. MCI older adults may not self-monitor conversational content well as compared to cognitively intact older adults. This could result in a larger proportion of words produced by participants (as opposed to the interviewers) during timed conversational sessions among the MCI older adults. Analyses of proportion of words would be a potentially useful and ecologically valid social marker that could be incorporated with other daily markers of everyday cognitive activity such as daily computer usage [8], walking speed and its variability [9], medication adherence [10, 11] time out of house [12] and others associated with early cognitive change that can be monitored over time by using passive and unobtrusive in-home sensing technologies [13]. In the present study, we compared the proportion of total word counts produced by participants among MCI and cognitively intact participants using the recorded conversations from the larger social engagement RCT noted above [5]. BODY.METHODS.PARTICIPANTS: Eighty-three older adults aged 70 years and older were enrolled and randomized into the intervention and control groups; mean age, 80.5 years and 76% female [5]. The original prevention study's inclusion and exclusion criteria are listed in Table 1. Out of forty-one participants assigned to the intervention group, 33 consented to allow their daily conversational intervention sessions to be transcribed for speech characteristics analyses (n=21 cognitively intact defined as Clinical Dementia Rating (CDR) [14]=0; n=12, MCI defined as CDR=0.5). Additionally, eight subjects (n=6 cognitively intact, n=2 MCI) recruited during a pilot-testing study who went through the same intervention protocol also consented and were included in this study, generating a total of 41 subjects reported here. As described above, the intervention group engaged in 30 to 35 minute semi-structured conversation sessions daily except weekends for six weeks with trained interviewers using Internet connected personal computers with a webcam. BODY.METHODS.CONVERSATION SESSION FORMAT: In order to take full advantage of a synthetic conversational format, we placed an emphasis on spontaneous responses rather than structured answers (i.e., the participants had to organize their thoughts). We used unstructured conversations such as talking about participants' "childhood memories", "hobbies", "siblings and parents", and "movies/books" [5] in addition to conversations generated by showing a daily picture stimuli (e.g., what is happening in this photo and where do you think it was taken?) and story generations (e.g., daily alphabetical conversation: tell me a story about apples, alligators, or alumni). These daily prompts were used uniformly for each session. We randomly selected one recorded session each from the baseline, the 3rd and 6th week of the trial, i.e., three sessions per participant. A single transcriber manually transcribed the conversations to extract word counts spoken by interviewers versus participants. This measure was implemented as one of the strategies to standardize the quality of interviews across interviewers, i.e. that one interviewer did not speak more than the others. Three interviewers were used in this trial. Each interviewer was assigned to talk with a specific participant during the 6 week trial. We assigned MCI and normal participants equally to each interviewer although interviewers were blind to the cognitive status (MCI vs. normal) of participants. BODY.METHODS.NEUROPSYCHOLOGICAL TESTS: In the original prevention study, pre- and post- trial changes in neuropsychological test scores were primary outcomes. In the current study, we examined the correlation between proportion of words spoken by participants and cognitive functions using the neuropsychological test scores at baseline. The cognitive tests (and cognitive domains they tap) used are as follows:(1) Immediate Memory: the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) Word List Learning [15]; (2) Delayed Memory: CERAD Word List Delayed Recall [15]; (3) Language: composite of verbal fluency for letters (F, A and S) [16]; (4) Psychomotor Speed: Trail Making A [17]; (5) Executive function: Trail Making B [17] and verbal fluency for category animals [16]; (6) Selective Attention/inhibition: Stroop test [16]; and (7) Pre-morbid and general intelligence: Wide Range Achievement Test-Revised (WRAT-R) [18]. BODY.METHODS.STATISTICAL ANALYSES: The proportion of words spoken by participants (out of total word counts in each trial session) was assessed. Student's t-test and Wilcoxon rank sum test were used to examine the differences in baseline characteristics and the relative proportion of words spoken between MCI and cognitively intact participants. Chi-square and Fisher's exact tests were used for categorical variables. We first assessed the spearman's correlation coefficients between the proportion of words spoken by participants and neuropsychological tests and demographic variables. Next, the relationship between cognitive status (MCI vs. intact) and the proportion of words spoken by participants was analyzed using a multivariate generalized estimating equation (GEE) model in order to account for multiple observations per subject. In the GEE model, participant age, gender, years of education, interviewer (creating 2 dummy variables distinguishing 3 interviewers) and assessment time point (creating 2 dummy variables indicating 3rd and 6th week of assessment, respectively) were included in the model. The assessment time point was included to examine if the interview order (baseline, week 3, week 6) showed any systematic differences. Second, we generated a logistic regression model using cognitive status as the outcome and proportion of word counts averaged over the three sessions as the predictor. We examined the Receiver Operating Characteristics Area under the Curve (ROC AUC) of distinguishing MCI subjects from those with intact cognition byproportion of word counts spoken by participants. Goodness of fits in GEE models and logistic regression models were examined through visual inspection of residuals and Hosmer-Lemeshow test, respectively. SAS 9.4 (Cary, NC, USA) was used for the analyses. BODY.RESULTS: Baseline characteristics of the 41 participants are listed in Table 2. Mean Mini-Mental State Examination (MMSE) [19] score at baseline was 26.9 (SD 2.1) among those with MCI; and 28.7 (SD 1.3) among those with normal cognition (p < .01). Category fluency test (semantic fluency, p=0.08) and letter fluency test (phonetic fluency, p=0.31) and stroop test scores (executive function/inhibition, p=0.07) were not significantly different between those with intact cognition and MCI, but other tests were. As for correlations, higher average proportion of words spoken by participants was correlated with older age (p=0.04) and fewer years of education (p=0.04). Among neuropsychological tests, MMSE (global cognition, p=0.02), word-list acquisition (learning, p=0.02) and stroop scores (executive function/inhibition, p=0.02) were negatively correlated with proportion of words spoken by participants. The average proportion of words spoken by participants across the 3 assessment time points was different between the groups (p=0.01) with 68.3% (SD 9.3) among those with MCI and 60.0% (SD 9.3) among cognitively intact. GEE analysis showed that those with MCI have about 6% higher proportion of word counts in a conversational session compared to those with intact cognition (Table 3). Older age (p=0.03) was also associated with higher proportion of word counts. There was no significant difference among assessment time points, although MCI subjects had a tendency of showing lower proportion of word counts at the 3rd and 6th week compared with baseline (i.e., more resembled to those observed among the normal, not in Table). Subjects interviewed by interviewer 2 talked significantly more (p<0.001), compared to those interviewed by interviewer 1, suggesting that Interviewer 2 could be more effective in getting the participants to talk, although we trained each interviewer extensively to standardize interviewer skills [5]. The logistic regression models showed the ROC AUC of identifying MCI (vs. normals) was 0.71 (95% Confidence Interval: 0.54 – 0.89, Odds Ratio for the proportion of word counts = 1.12, p=0.02) when average proportion of word counts spoken by subjects was included univariately into the model. Adding age and education in the model further improved the ROC AUC to 0.84 (95% Confidence Interval: 0.69 – 0.99). BODY.DISCUSSION: Holding conversations involves a synthesis of social and cognitive functions; a person is required to actively construct a representation of what another person is thinking or feeling, what that person believes, what they desire, and what their perspective is on topics and ideas [20, 21]. All of this "social cognition" requires remembering what was said a few seconds or minutes before, planning and organizing one's next thoughts, keeping track of the conversation, iterating and updating the interaction, as well asself-monitoring in order to take turns, not interrupt, understand another's feelings and inhibit inappropriate behaviors. Thus, at various times, attention, executive function, inhibition, abstract reasoning, memory and language abilities are simultaneously engaged. We compared proportion of word counts generated by participants out of total word counts, rather than comparing the word counts per se because our interest was in how two people (participants and interviewers) interact with each other in one-on-one conversations and in order to control for total duration of conversational sessions. We found that MCI participants spoke a greater proportion of words during the timed remote video telecommunication conversational sessions than those with normal cognitive function. Past research has examined the complexity, content and acoustic features of spoken language as an indicator of aging or cognitive status by using answers to neuropsychological tests or to specific tasks (e.g., ask subjects to reada paragraph, speech provided in press conferences) [2, 22, 23] To our knowledge, this is the first study attempting to examine interactions between two peoplein a one-on-one conversational setting using the proportion of word counts of MCI in comparison with normal participants. Using the Alzheimer's Disease Neuroimaging Initiative (ADNI) data, discriminatory ability (indicated by the area under the cure (AUC) of the receiver operator curve (ROC)) of biomarkers together with age and gender information in distinguishing MCI from the normal has been reported to range from 0.68 (hippocampal volume, age and gender in the model) to 0.77 (CSF Aβ42, CSF tau, sex and age in the model) [24].Others have found somewhat higher AUC values ranging from 0.70 to 0.96 for fluid biomarkers which may relate to differing methods or characteristics of the samples studied [25-27]. The ROC AUCs in our study using word counts (0.71 with the proportion of word counts alone and 0.84 with the word counts, age and education information) were comparable to many of the currently used biomarkers in the field. Thus we highlight that using the quantitative behavioral biomarker of word counts is particularly promising as an approach to identify populations at risk for decline or to track response to therapies, especially considering that the data collection can be done at home, and does not require invasive or expensive biomarker assessments. Neuropsychological criteria used to define MCI [28] includes objective evidence of impairment on at least two tests within four or more cognitive domains, with scores falling at least 1 standard deviation (SD) or more below age-stratified normative data. The language domain is commonly measured by tests such as the Boston Naming Test [29], letter fluency(phonemic fluency) and category fluency tests (semantic fluency) [16]. In Alzheimer's disease (AD), category fluency has generally been found to be disproportionately impaired, whereas letter fluency ability is less impaired [30, 31], although not all studies agree with this finding [32]. It is hypothesized that the disproportionate impairment in semantic fluency, as opposed to phonemic fluency, could occur because the former relies more on temporal-lobe semantic stores, the area which is affected by AD. Our finding that participants with MCI (the prodromal stage of AD) spoke a higher proportion of word counts in free conversations may be reflecting their subtle decline in semantic fluency abilities. Patients with dementia are known to have high incidence of circumlocutions (i.e., the use of many words where fewer would do) and semantic jargon in their spontaneous speech [33]. We found a somewhat larger difference between MCI and normal groups on a semantic fluency test (p=0.08) than on a phonemic fluency test (p=0.31), although neither difference was statistically significant, likely due to small sample size. Possibly MCI participants may tend to struggle to find the right words and therefore may be more likely to need to substitute words in the conversation to convey their thoughts, especially in the early stage of MCI when phonemic fluency is still preserved, leading to increased proportion of word counts in timed conversations. It is noteworthy that in our study the differences insemantic and phonemic test scores between the MCI participants and those with intact cognition were not significant, while the proportion of words spoken by participants showed a significant difference between the two groups. The latter measure may be more sensitive to cognitive decline than traditional neuropsychological tests and may identify early stage MCI before impairment becomes symptomatic through neuropsychological tests. There are other possible underlying mechanisms that may explain the disproportionate proportion of words contributed to a conversation by MCI participants. Increased word counts among MCI individuals may be due to subtle difficulties with the executive and self-monitoring aspects of conversation. Also MCI participants could have reduced passage-of-time estimation abilities relative to those with normal cognitive function. Thus those with MCI would not be able to anticipate and prepare for the end of the sessions. However, there is some evidence that time estimation is not affected in MCI, although it is associated with aging [34]. There is some evidence that MCI participants may acquire deficits in social cognition such as the ability to make inferences about the cognitive state, emotions and intentions of other people [35, 36]. Misreading of social cues and intent during social exchanges could result in more discursive conversation among MCI participants. Epidemiological evidence suggests that tracking changes in linguistic features may be a promising tool in identifying early signs of AD. The Nun Study found a link between linguistic density (e.g., complexity, vivacity, fluency) in early life (most were in their 20s) and the risk of developing Alzheimer's disease later on, by examining autobiographical essays written by the nuns upon joining the Sisterhood [37]. Other studies also suggest that subtle language impairment occurs long before an MCI diagnosis or at a very early stage of MCI. For example, Oulhaji et al., [38] showed that using the CAMCOG (the Cambridge Cognitive Examination), verbal expression and learning scores at baseline, conducted about 20 years beforehand, were the most significant predictors of incident MCI. Amieva et al., [39] showed that low category fluency test scores can be seen nine years before the clinical diagnosis of Alzheimer's disease. Recent MRI studies have found associations between the size and complexity of real-world social networks and the density of grey matter [3] and amygdala volume [40], providing some support for a link between biomarkers (such as brain structure) and social network size, an indirect measure of how well subjects communicate with others. Research is increasingly focused on identifying AD at the MCI stage, or even earlier, in order to prompt interventions and to access clinical trials with disease-modifying drugs [41]. Clearly, higher-order functional activities have been shown to distinguish those at risk of developing MCI from those remaining cognitively normal [42-44]. Studies utilizing continuous, unobtrusively monitored in-home activity data in a community setting also showed that these ecologically valid activity measures could identify early changes among those who convert to MCI [13]. We have been following seniors at their home over 4 years recording their in-home activities unobtrusively, including decline in computer use at home [8], changes in in-home walking speed and variability [9], and time out of house as an indicator of the amount of social interactions. Current information analysis science can provide objective assessments of the nature of social interactions through the examination of speech characteristics, including sentence complexity, verbal fluency, vocabulary, and acoustic features including affect which was not possible a decade ago [1, 2]. It will take some time to realize the assessment of speech characteristics including developing automatized algorithms to protect privacy, developing a reliable way of recording conversations, storing or transmitting the recorded data for analysis. However, the assessment protocol could be implemented as one of the in-home monitored activities to enhance the prediction of those at-risk of cognitive impairment in communities in the future. The results presented in this report, together with the past observational studies mentioned above, suggest that examining the way older adults interact with each other in conversation could facilitate the development of powerful tools for identifying early signs of MCI in community dwelling older adults. Further studies are required to validate our findings and to create practical applications of assessment protocols. There were some limitations to this study. The sample size was relatively small. Participants were volunteers interested in participating in a behavioral intervention trial and this might limit the generalize ability of the results. The conversation session format used in this study was a remote telecommunications video chat between participants at their home and trained interviewers at the study site. Video chat conversational format differs in some ways from face to face conversation. However, we paid special attention to creating a user-friendly environment, including touch-screen monitors, which eliminated mouse use when receiving calls, a large monitor that allowed eye-to-eye contact during in-person conversations in order to retain attention during the session, and pop-up pictures on the screen to evoke conversations without any effort by the participants. Additionally, previous studies have shown that remote telehealth communication formats can be effective for a variety of assessments and interventions with older adults [45]. BODY.CONCLUSION: An ecologically valid social marker such as the proportion of spoken words produced during free conversation may be sensitive to transitions from normal cognition to MCI and may be able to detect the transitions before any changes in conventional cognitive tests. It could complement biomarker studies in selecting or identifying at-risk participants during their pre-symptomatic phase. Examinations of interactions among subjects in free conversational speech could provide an additional area of research useful for the early identification of MCI.

TITLE: The display effects of patients’ self-assessment on traumatic acute pain on the proportion and timing of analgesics administration in the emergency department ABSTRACT.BACKGROUND: Acute pain assessment in the emergency department (ED) is important in particular during the triage process. Early pain assessment and management improve outcome. The objective of this study was to determine the effects of documentation and display of patient's self-assessment of pain using numerical rating scale (NRS) on analgesic use among adult trauma patients in ED. ABSTRACT.METHODS: A randomized control trial was conducted recruiting 216 trauma patients who presented to ED of two tertiary centers. Pain score was done using NRS for all patients. They were randomized into pain score display group or not displayed in the control. The outcome measured were proportion of patients receiving analgesics and timing from triage to analgesic administration. ABSTRACT.RESULTS: The proportion of patients who received analgesics when pain score was displayed was 6.5% more than when pain score was not displayed. This difference was however not statistically significant. However, stratified categorical analysis using chi-square showed that the displayed severe pain group was 1.3 times more likely to receive analgesics compared to the non-displayed group. The mean timing to analgesic administration for the displayed and non-displayed groups were 81.3 ± 41.2 (95% C.I 65.9, 96.7) and 88.7 ± 45.4 (95% C.I 69.0, 108.3), respectively (p > 0.05). ABSTRACT.CONCLUSIONS: The proportion of patients who received analgesics increased when NRS was displayed. However, the pain display has no significant effect on the timing of analgesics. BODY.1 BACKGROUND: The usual scenario in the emergency department (ED) is an overworked doctor, carefully putting history together, examination, and investigations, to arrive at a diagnosis. This is often done with much time constraints and poor attention paid to acute pain management. However, a physician's primary duty is to comfort, manage, and reduce the suffering of patients. This study attempted to address this contradiction, by introducing a simple intervention in the ED. This is an effort to evaluate the effect of displaying pain score, without any other intervention, on analgesic use. As pain score is recommended as a vital sign in many international protocols, it is in a way asking if we can improve acute pain management if we implement the pain score display at triaging in the ED. BODY.2 OBJECTIVES: The main aim of this study is to determine the effect of documentation and display of patient self-assessment of pain using numerical rating scale (NRS) on analgesic administration. Specifically, we are looking into the effects of numerical pain score display on the proportion of patients receiving analgesics and the timing of analgesic use. BODY.3 METHODS: This was a cross-sectional randomization study in the ED of two tertiary teaching hospitals with an average annual patient attendance of 140,000. This study was granted ethical approval by the research and ethical committee of the institution. The following were the inclusion criteria: (a) Adult ≥ 20 years old (b) Patients complaining of acute pain secondary to trauma (c) Patients triaged to non-critical zone only (d) GCS 15/15 The following were the exclusion criteria: (a) Patients with language barrier (b) Patients who were intoxicated (c) Patients with altered mental status or psychiatric illness (d) Patient under police custody (e) Patients with potentially life-threatening diseases or injuries (f) Non-traumatic causes of acute pain Patients were carefully selected among whom who have likelihood of presenting with significant acute pain secondary to trauma, while avoiding interference with evaluation and care of patients with potentially life-threatening illness or injury. All patients attending the ED would be triaged by a triage officer at the entrance of the department. Critical patients would be admitted to the resuscitation bay immediately after the triage decision had been made. The semi-critical and non-critical patients would be triaged further including the pain scoring assessment. Acute pain assessment by using NRS is a part of a vital sign monitoring parameter included into the triage protocol of the department. Patients would be given analgesics at the triage counter if they presented with moderate to severe pain. Patients would be up-triaged and admitted to the treatment cubicle if they developed worsening of pain while waiting to be seen by doctors. Only patients who were able to give consent and had a clear mind to understand and do pain assessment were included in the study. Randomization was done prior to selection of patients. Patients were assigned to display pain score and non-display pain score groups, using a computer-generated randomization method, in blocks of four. These randomized selections were sealed in opaque brown, non-transparent, numbered envelop and arranged in ascending order. The envelopes were opened only after pain assessment was done and documented by researcher. The researcher and the triage officer were blinded to the randomization while doing the pain score and initial data collection. This was important to avoid bias when pain score was being performed. Verbal consent was obtained from each patient. The sample size was calculated by using the following parameters:(1)Po=0.5(2)Pi=0.3where α = 0.05 (95% CI), study power = 0.8 (80% ), two proportion samples Po = 0.5 and Pi = 0.3, and M = 1. The sample size required were 220 patients (110 per arm) including the 10% dropout. BODY.3 METHODS.3.1 DATA COLLECTION: The NRS was used as the pain scale assessment tool as this has been assessed as the best tool to be used among trauma patients in the ED. (Figure 1) [1]. A bold red marker pen was used to display the pain score. A yellow sticker measuring 10 × 7 cm was used to attach the pain score at the top right hand corner of the front cover of medical case notes. This pain score was displayed prominently in the patients' medical clerk sheet in one group and not displayed in another group. Figure 1Numerical rating scale (NRS). Pain assessment was done at triage after chief complaint and vital signs were assessed. This was done by the patients, without relying on physicians' impression. This is as suggested by the Canadian Association of Emergency Physicians (CAEP) consensus document on emergency pain management [2]. Instructions on how to do a pain score was explained to the patients by the researcher. Instruction was given by using local languages and dialects. Relatives were discouraged from translating or trying to answer for the patient. The numerical rating scale was shown to the patient. Patients were asked to give pain score value according to their own assessment of pain severity. They were excluded from the study if they did not understand the instructions after being repeated twice. The data was entered into the data collection form. Based on the previous study carried out by Silka et al., we decided that the NRS score of 7 to 10 is severe pain, NRS score of 4 to 6 is clinically significant moderate pain, and any score of 3 and below is mild pain. Data entry and analysis were done using Statistical Packages for Social Science (SPSS) version 12.0.1 software which was registered by the institution. Numerical pain score was converted to categorical variable. Chi-square test was used in the analysis of the differences of analgesic administration between the displayed pain score and not displayed pain score groups. The confounding effect of pain severity on this relationship was analyzed by using Mantel-Hanszel estimate. Timing to analgesics was presented as mean and analyzed by using ANOVA test. Statistical significance was considered at p value of less than 0.05. Data exploration was done using descriptive statistics and presented as charts and tables for each variable. BODY.4 RESULTS: The number of patients who met all criteria was 216. Finally, there were 107 patients (50.5%) in the study group and 99 (49.5%) patients in the control group after exclusion due to various reasons. The general demographic data comparison between the two study groups is as shown in Table 1. There is no significant difference in the gender distribution, age group, education, mechanism of injury, and pain score between the study groups (p > 0.05). Majority of patients who presented with acute pain in this study group were in the range of 20 to 29 years of age (n = 99, 49.5%). The distribution of the pain score groupings in the displayed NRS group for the mild, moderate, and severe pain are 12.9% (n = 13), 46.5% (n = 47), and 40.6% (n = 41), respectively. Likewise, the distribution of the pain score groupings in the non-displayed NRS group for the mild, moderate, and severe pain are 17.2% (n = 17), 49.5% (n = 49), and 33.3% (n = 33), respectively. The proportion of patients who received parenteral analgesics for mild, moderate, and severe pain in displayed group was 7.7%, 21.7%, and 47.6%, respectively (p < 0.05). Likewise, there is an increment of proportion who received analgesics in the non-displayed group from 23.5% to 31.3% with increasing pain severity (p < 0.05). The proportion of patients who received analgesics when pain score was displayed was 6.5% more than when pain score was not displayed. This difference was however not statistically significant (p = 0.30) (Figure 2 and Table 2). Table 1 Demographic comparison between the study and control groups Variables Non-display (control) group ( n   = 99) Display (trial) group ( n   = 107) p value Age in years (mean) 35.6 38.6 0.639 Gender distribution ( n  = Male or Female) Male = 70 (70.1%) Male = 73 (68.2%) 0.242 Female = 29 (29.2%) Female = 34 (31.8%) Education at minimum secondary school level 59 (59.6%) 64 (59.8%) 0.662 Mean (s.d) pain score (NRS) 5.7 (1.9) 6.1 (2.6) 0.300 Severe pain score (more than 7) ( n ) 33 (33.3%) 41 (38.3%) 0.492 Motor vehicle crash patients ( n ) 51 (51.5%) 42 (39.3%) 0.621 Patients received intravenous analgesics ( n ) 49 (49.5%) 58 (54.2%) 0.310 Figure 2Bar chart showing percentage of patients who received analgesics in trial versus control group. Table 2 Odd ratio of patients receiving analgesics   Crude odd ratio Weighted odd ratio p value Displayed pain score × Receiving analgesics 1.396   0.30 Pain group a displayed pain score × Receiving analgesics   1.306 0.42 a Confounder effect of variable (confounder effect of pain severity on displayed pain score was analyzed using Mantel-Hanszel estimate). The mean timing (in minutes) to analgesic administration for the displayed and non-displayed groups were 81.3 ± 41.2 (95% C.I 65.9, 96.7) and 88.7 ± 45.4 (95% C.I 69.0, 108.3), respectively (p = 0.538). The distribution of timing of analgesics according to the severity of pain is as shown in Table 3. Table 3 Test results of analysis of timing to analgesics by displayed stratified Pain Score using ANOVA Display pain score Mean (minutes) Std. deviation 95% CI (upper, lower) Minimum Maximum Pain score 4 to 6 (displayed) 84.2 51.8 16.4, 47.2 20.0 190.0 Pain score 4 to 6 (not displayed) 89.6 40.4 55.8, 123.4 25.0 170.0 Pain score > 6 (displayed) 80.8 36.0 8.0, 63.9 20.0 165.0 Pain score > 6 (not displayed) 94.3 48.6 72.7, 96.3 25.0 174.0 p  = 0.946. BODY.5 DISCUSSION: Painful suffering is neither a diagnosis nor a disease. Nevertheless, it is a condition experienced by patients posttrauma that needs to be attended to attentively. Over the last few years, there has been much progress in the understanding of the pathophysiology of pain and its effect on disease and healing. More importantly, evidence has emerged on the detrimental effect of neglecting pain on disease progression and patient recovery [3],[4]. There have been many attempts to introduce ways to improve pain management in the ED. Great steps to improving pain and suffering in a patient start with understanding what pain means to the patient. Pain is what the patient states it is. Physicians must respect this. A diverse spectrum of psychological, sociocultural, temporal, and situational variables affects how people perceive and express their pain [5],[6]. The patients' self-report is the most reliable indicator of the presence and intensity of pain. Health care professionals often fail to routinely assess and document pain. Physicians should trust the patients' subjective reports of pain unless there is evidence to the contrary [7],[8]. Few studies had shown the positive effects of pain score display on analgesic use in the ED. In a retrospective study by Nelson, the effect of introducing a mandated verbal numeric pain scale on the incidence and timing of analgesic administration in trauma and non-trauma patients in the ED was studied. Analgesic use increased from 25% to 36%, and analgesics were administered more rapidly after the scale was introduced [9],[10]. Considering the positive results shown by these studies, it was felt that a randomized control study to determine the effect of documentation and display of patient self-assessment of pain using NRS, on analgesic administration was an important move to increase the evidence of the importance of pain score as a fifth vital sign in triage. In this study, a detailed analysis of the three different pain score groups, minimal, moderate, and severe pain, has shown that with the increasing pain score, more patients received analgesics. This is more frequent in the displayed pain score group. In Silka's study in 2004, no patient who reported mild pain received analgesics, whereas 72% of patients with moderate to severe pain did receive analgesics. A similar study was done in 2004 by Thomas. That study used the visual analog scale (VAS), which was displayed at the patient's ED chart and placed at the head of the ED stretcher. The study by Thomas showed that 63% of patients in the group with graphical display of VAS score received analgesics compared to 58.7% in tabulation group and 55.7% in the control group [11],[12]. Our study has found that analgesic administration is significantly associated with increasing severity of pain. This study gives the benefit of knowing what the pain score was in all patients. In patients where pain score was not displayed, a significant proportion of patients with moderate and severe pain still received analgesics. This shows that there are other factors influencing the attending medical officer to prescribe analgesics. Clinical judgment, the doctors' own assessment of pain, seems to have played a significant role here. What can be concluded here is that more patients received analgesics with increasing severity of pain, whether the pain score was displayed or not. It is not only the awareness of a displayed pain score that increased the likelihood of analgesics but also the clinical judgment of the presence of pain is an important factor. However, in this study, the displayed pain score did not affect the timing to analgesics compared to when pain score was not displayed. The study by Silka also showed no difference in the timing to first analgesics when comparing those patients with or without VPS. Pain assessment using NRS at the time of triage and prominent display of the pain score in patient medical record did not alter the timeliness of analgesic administration. Time to analgesics has not been studied in the ED in this country previously as far as the author's awareness. There is no nationwide system currently in place to triage a patient with a high pain score to see the doctor earlier. There are also no guidelines to administer analgesics earlier in patients with high pain score, while they wait for doctors. These are the measures that could be introduced to reduce the time the patient is in pain while waiting for analgesic administration. BODY.5 DISCUSSION.5.1 LIMITATIONS: The main limitation of this study is the practice of analgesic administration in the ED being influenced by the awareness that the study is going on. The presence of the researcher in the triage and the new sticky label on the medical case notes with the pain score developed the awareness that they were being monitored. Perhaps this created some pressure to give more analgesia than usual or otherwise. This inevitable Hawthorne effect was counterbalanced by the utilization of a control group. Notably, patients and physician were blinded to the study null hypothesis. Only patient factors that influenced pain score and analgesic administration were studied. Factors regarding the doctors' experience or knowledge of pain management were not analyzed. These factors could not be studied as there was a high turnover of doctors in the ED and many different doctors work for the three shifts. Other factors that may influence analgesic administration include the number of doctors on duty and the number of patients on the days of study or particular shift. If there were many patients and the doctors were very busy, they might ignore the pain score and try to work fast in their limited time per patient. The time of the day when the study was conducted may also affect the outcome. These were not considered in the study. BODY.6 CONCLUSIONS: This study has determined the effect of documentation and display of patient self-assessment of pain using NRS on analgesic administration. The proportion of patients who received analgesics increased when NRS was displayed. However, the pain display has no significant effect on the timing of analgesics. BODY.COMPETING INTERESTS: The authors declare that they have no competing interests. BODY.AUTHORS’ CONTRIBUTIONS: NHR involved in the conception of the study and preparing the manuscript. CA involved in data collection and statistical analysis. Both authors read and approved the final manuscript.

TITLE: Non-Invasive Brain Stimulation for Children with Autism Spectrum Disorders: A Short-Term Outcome Study ABSTRACT: Non-Invasive Brain Stimulation (NIBS) is a relatively new therapeutic approach that has shown beneficial effects in Autism Spectrum Disorder (ASD). One question to be answered is how enduring its neuromodulatory effect could be. Twenty-four patients with ASD (mean age: 12.2 years) received 20 sessions of NIBS over the left dorsolateral prefrontal cortex (L-DLPFC). They were randomized into two groups with two (G1) or three (G2) clinical evaluations before NIBS. Both groups had a complete follow-up at six months after the intervention, with the aim of determining the short-term outcome using the total score on the Autism Behavior Checklist, Autism Treatment Evaluation Checklist, and the Autism Diagnostic Interview. Transcranial Direct Current Stimulation (tDCS) was used in ASD patients aged <11 years, and repetitive Transcranial Magnetic Stimulation (rTMS) for 11–13-year-olds. Observation points were at one, three, and six months after completing all the sessions of NIBS. A significant reduction in the total score on the three clinical scales was observed and maintained during the first six months after treatment, with a slight and non-significant tendency to increase the scores in the last evaluation. Twenty sessions of NIBS over the L-DLPFC improves autistic symptoms in ASD children, with a lasting effect of six months. BODY.1. INTRODUCTION: Autism Spectrum Disorders (ASD) still pose challenges for neuroscience for many reasons; one of these is how to apply the advances in neuroscience to understanding and treating ASD by improving diagnosis, interventions, and treatments. To date, diagnosis is made on clinical bases in the absence of a true biomarker for diagnosis and a better therapeutic approach. Different pharmacological and non-pharmacological therapies have shown positive results but are far from leading to a significant improvement in the core symptoms of patients with ASD. A high incidence of brain anomalies had been described by different methods in autistic patients in comparison with neurotypical patients, including MRI techniques and anatomic studies [1,2,3]. One interesting feature described by Casanova et al. was the abnormal structure in cortical mini-columns, with poor neuropil development in ASD patients, which probably accounts for intracortical inhibitory dysfunction [1,4]. Alterations in the GABAergic signaling pathway may characterize autistic neurobiology, which seems to not simply be related to a decreased GABA concentration, but probably to perturbations in key components of the GABAergic pathway beyond GABA levels, such as receptors and inhibitory neuronal density [5]. There is also some research exploring functional intracortical inhibition in motor areas of ASD patients that reinforce these ideas; nevertheless, its functional expression in terms of modulation by means of paired pulse stimulation protocol with TMS is really less remarkable than was expected, and it has been demonstrated in only a fraction of the ASD population [6]. In other experiments, indirect evidence has been described related to an aberrant plasticity based on an imbalanced excitatory and inhibitory cortical tone as well as biased GABAergic dysfunction [7]. Casanova et al. were the first to propose the use of Non-Invasive Brain Stimulation (NIBS) for ASD patients, based on the possibility that low-frequency repetitive TMS could somehow improve GABAergic neurotransmission; in their case, low-frequency repetitive Transcranial Magnetic Stimulation (rTMS) was used as a way to improve intracortical inhibition in ASD patients. They applied one weekly session over 12 weeks in a group of 25 ASD patients and described an increase in gamma activity in the EEG evoked by a visual processing paradigm, with changes in Event Related Potential (ERP), improved error monitoring, and correction function in a visual recognition task after the intervention [8,9]. There are just a few other studies providing evidence of the potential efficacy of both rTMS and transcranial direct current stimulation (tDCS) in ASD (excluding case reports). Unfortunately, most of the trials are limited to a low number of sessions (not more than 15 in the best-case scenario), and it was not known how enduring the modulatory effect was [10,11,12,13]. Dorsolateral prefrontal cortex (DLPFC) is the preferred target for many other clinical conditions, thus also in ASD based on its connections with other cortical and subcortical structures. Nevertheless, other considerations are valid about the feasibility for the selection of other targets [1,14]. Studies in ASD patients using low-frequency rTMS over the left DLPFC (L-DLPFC) have shown a significant improvement in some components of event-related cortical potentials (N200 and P300) as well as a significant reduction in response errors during cognitive tasks, repetitive behavior, and irritability [9]. Other authors using cathodal tDCS over the L-DLPFC have also reported beneficial effects for ASD in young adults. Curiously, there is also a published work of a trial with anodal stimulation over DLPFC reporting improvement in autistic symptomatology [10,11]. If we accept the hypothesis that an intra-cortical inhibitory dysfunction exists in ASD patients that can contribute to several dysfunctions in the motor, sensorial, and cognitive domains, we can expect some improvement in autistic behavior after applying brain-stimulating protocols that may potentiate intracortical inhibition, probably increasing the amount of available endogenous GABA in neuronal networks related to the stimulation target. In previous experiments, low-frequency rTMS and cathodal stimulation over the left DLPFC in children with ASD were associated with an improvement in autistic behavior one week after completing 20 sessions of transcranial stimulation [15,16]. One important question is how lasting this effect would be; in the present study, we describe the short-term clinical response during the first six months after treatment. We hypothesized that 20 sessions of NIBS over the L-DLPFC are expected to have a lasting positive neuromodulatory effect in patients with ASD. BODY.2. MATERIALS AND METHODS.2.1. STUDY DESIGN: A controlled, randomized, and partial crossover trial was carried out in 24 children with ASD. Fifteen patients received the intervention after two clinical evaluations within one month (group 1, G1); nine patients started receiving the intervention after two months with three evaluation points at one-month intervals (group 2, G2). We assume that according to the stimulation protocol to be used (one daily session, up to a total of 20), it would be difficult to determine a washout period and patients from G2 ran in parallel to patients from G1 while they were receiving the intervention. BODY.2. MATERIALS AND METHODS.2.2. SAMPLE SELECTION AND GROUP DISTRIBUTION: Children with ASD were recruited from the ambulatory service of the International Center for Neurological Restoration and Marfán-Borrás Hospital, from November 2015 to October 2016. Their diagnosis was made based on DSM-V diagnostic criteria, with the consensus of a multidisciplinary team including two Child Psychiatrists and two Neuropediatric Specialists. Only patients with a slight or moderate grade of severity were included according to their clinical characteristics, with stability during the last two months, and no changes in their therapeutic scheme in either pharmacologic or non-pharmacologic interventions. Diagnosis was confirmed by the results of the Childhood Autism Rating Scale (CARS) [17] and the Autism Diagnostic Interview, Revisited Edition (ADI-R, diagnostic algorithm) [18]. Patients with severe autistic behavior were excluded, as well as patients with concurrent epilepsy. Patients with unclear diagnosis or with diagnostic disagreement between neurologist and psychiatrist opinion were also excluded. If any change was needed in their therapeutic scheme, the patient was also excluded from the trial. We applied a simple randomization technique to assign patients to G1 (early intervention group, after one month of follow-up) or G2 (intervention after two months of follow-up). BODY.2. MATERIALS AND METHODS.2.3. CLINICAL EVALUATION: Three main clinical scales were used as outcome measurements for all patients, according to their parent opinions: ADI-R (algorithm for current condition), the Autism Behavioral Checklist (ABC) [19], and the Autism Treatment Evaluation Checklist (ATEC) [20]. All the scales were applied by a Child Psychiatrist who was not involved in the intervention, and patients were evaluated at one, three, and six months after completing the 20 sessions. The Global Clinical Impression Scale (GCIS) [21] was also applied as a complementary, more qualitative evaluation. The Wilcoxon matched pair test and Mann–Whitney U test were used either for intra- or between-group comparisons at the different evaluation moments (α = 0.05). BODY.2. MATERIALS AND METHODS.2.4. NEUROPHYSIOLOGICAL EVALUATION.2.4.1. FUNCTIONAL BRAIN CONNECTIVITY: An electroencephalogram (EEG) based connectivity analysis was carried out in all the ASD patients, but only 15 good-quality EEG recording were obtained for connectivity analysis (nine from the tDCS group and six from the rTMS group). EEG traces taken one week before starting the intervention and one week after finishing it were analyzed. Significant windows were selected from the EEG trace in open eyes state (38 from each patient), because they were more often artifact-free than the closed-eyes state. We used a 19-electrode montage including Fp1, Fp2, F7, F8, F3, F4, C3, C4, T5, T6, T3, T4, P3, P4, O1, O2, Fz, Cz, and Pz, from the 10/20 system. Electrode Impedance was kept below 5 kΩ. Functional connectivity was analyzed based on the synchronization likelihood between electrodes for the five frequency bands: δ (1–3.9 Hz), θ (4–7.9 Hz) α (8–12.9 Hz), β (13–29.9 Hz), and γ (30–35 Hz) [22]. Mathematical analysis was developed with connectivity algorithms implemented in MATLAB v.7.7 R2008b. Significant connectivity (p < 0.05) in each frequency band was represented on an X/Y coordinate according to the 10/20 system for each brain stimulation modality independently, and the results by group were represented over the scalp surface map. BODY.2. MATERIALS AND METHODS.2.4. NEUROPHYSIOLOGICAL EVALUATION.2.4.2. EVENT-RELATED POTENTIALS: Only in six children was it possible to carry out a passive oddball paradigm for P300 Event-Related Potentials (ERPs) before the intervention; four of them received tDCS and two rTMS. Patients were seated in a sound- and light-attenuated room while a paradigm was conducted consisting of 200 stimuli, 80% frequent (500 Hz) and 20% infrequent target (1000 Hz) tones, delivered through headphones while children were watching a silent movie. In the passive version of the oddball task the subject ́s attention is usually directed away from the sequence of standard and deviant tones toward another, moderately demanding task, usually in a different modality [23]. We proposed a version of this paradigm considering the characteristics of the autistic children, with poor collaboration. All stimuli (50 ms; 5 ms rise and fall time) were presented binaurally with an inter-stimulus interval of 1300 ms. To record the auditory P300 ERPs, 19 surface electrodes (Ag/ClAg) were attached to the scalp according to the international electrode placement 10–20 system (Fp1, Fp2, F3, F4, F7, F8, C3, C4, T3, T4, T5, T6, P3, P4, O1, O2, Fz, Cz, Pz), with additional electrodes for EOG (lateral to the outer canthus of the right eye and above the middle of the left eyebrow), and referenced to the linked earlobe. The electrode impedance was kept below 5 kΩ. EEG data were off-line averaged (100 ms prior and 700 ms after stimulus onset), and P300 latency and amplitude were quantified at Fz, Cz, and Pz. A continuous acquisition system was employed (Medicid 5, Neuronic SA, Cuba) and EEG data were EOG-corrected offline. The sampling rate of all channels was 200 Hz. To assure auditory system normality, Auditory Brainstem Response was previously recorded. The stimuli were 0.1 ms alternating clicks delivered through a headphone (DR-531B-7, Elegas Acous Co. Ltd., Tokyo, Japan). The records were obtained using the evoked potentials measuring system Neuropack M1 (Nihon Kohden, Tokyo, Japan). P300 ERPs were obtained before and after NIBS, and P300 latency and amplitude values from the two evaluations were compared (from Fz electrode position; Wilcoxon matched pairs test, α < 0.05). All measurements were of the difference wave (infrequent wave minus frequent wave) and in this case, due to the low number of patients' recordable P300, the analysis was performed considering all patients as a single group. BODY.2. MATERIALS AND METHODS.2.5. INTERVENTION: Patients received one daily session of NIBS, from Monday to Friday, for a total of 20 sessions. tDCS was used in patients 10 years old or younger, and rTMS in patients 11 and older. The reason for using tDCS in younger children instead of rTMS was that in this case a major degree of collaboration is needed to assure effective focal stimulation over the selected target (left dorsolateral prefrontal cortex, L-DLPFC). During the stimulation sessions patients, were comfortably seated while watching TV cartoons of their preference or listening to music and playing with small, simple toys. BODY.2. MATERIALS AND METHODS.2.5. INTERVENTION.2.5.1. TRANSCRANIAL DIRECT CURRENT STIMULATION (TDCS): tDCS was only used in patients 10 years old or younger (Neuroconn tDCS stimulator, München, Germany). A cathode was positioned over F3 (10/20 international EEG electrode system), and an anode over the proximal right arm. The stimulation intensity was 1 mA, and was maintained for 20 min. Each electrode pad was humidified with a 0.9% NaCl solution. BODY.2. MATERIALS AND METHODS.2.5. INTERVENTION.2.5.2. REPETITIVE TRANSCRANIAL MAGNETIC STIMULATION (RTMS): rTMS was used in patients older than 10 years and 11 months (MagStim Rapid2, Whitland, UK). A butterfly coil with air cooling system (MagStim Double 70 mm Air Film Coil) was used. The center of the coil was located over F3, back handed 45° from the midline, and a total of 1500 pulses were delivered in each session, at 1 Hz of frequency and an intensity of 90% of the resting motor threshold (i.e., the minimum stimulation intensity required to elicit a discernible hand muscle response in at least three of five consecutive pulses) [24]. Sessions were subdivided into four trains of 375 pulses, with a 1-min interval between each allowing for free movement of the head and neck. Most of the patients rejected the use of earplugs, and in the best-case scenario they agreed to listen to music by wearing headphones from their own devices. BODY.2. MATERIALS AND METHODS.2.6. ETHICAL CONSIDERATIONS: All the procedures followed the rules of the Declaration of Helsinki of 1975 for human research, and the study was approved by the scientific and ethics committee from the International Center for Neurological Restoration (CIREN37/2015). Parents gave written informed consent for their children to be considered for inclusion in the study, and where possible children also gave their consent. BODY.3. RESULTS.3.1. CHANGE IN CLINICAL SCALES ONE MONTH AFTER THE INTERVENTION: As a global result (G1 + G2), a significant decrease in the total score was observed in ADI-R, ABC, and ATEC scales one month after the intervention (Wilcoxon matched pair test; ABC, Z = 3.823, p = 0.000131; ADI-R, 3.337, p = 0.000846; ATEC, Z = 3.723, p = 0.000196). Figure 1a represents the global scores for the three main clinical scales we applied for the evaluation of autism behavior. The values are in correspondence with qualitative changes described by their relative uniqueness in socialization and communication domains, and with the GCIS (pre-intervention: 3.47 ± 0.6; post-intervention: 2.95 ± 0.2. Wilcoxon pair series test: Z = 2.803, p = 0.005062). All of the patients' autistic behavior improved according to the scale results observed one month after the intervention. A comparison between the change in the total score of clinical scales did not show any significant differences correlated with the use of tDCS and rTMS (Mann–Whitney U Test, p > 0.05). Initial evaluation did not show any differences in the total score between groups when we looked for any evidence of age-dependent characteristics in both groups. The clinical response to NIBS was apparently independent of group age and type of intervention (See Figure 1b). Figure 2 shows that no significant differences were observed between G1 and G2 in the initial clinical scale scores before the intervention (Mann–Whitney U test, p > 0.05); but values observed in G1 after NIBS showed a very important change when compared with the initial values (Wilcoxon; ABC, Z = 3.823, p = 0.000132; ADI-R, Z = 3.550, p = 0.000385 ;ATEC, Z = 5.526, p = 0.000241); the same behavior was seen in G2 after the intervention (Wilcoxon; ABC, Z = 3.295, p = 0.000982; ADI-R, Z = 3.588, p = 0.009633; ATEC, Z = 3.179, p = 0.001474). BODY.3. RESULTS.3.2. CHANGE IN CLINICAL SCALES DURING THE FIRST SIX MONTHS AFTER THE INTERVENTION: Post-intervention follow-up was extended over six months. For both groups G1 and G2, NIBS induced a significant change in clinical scale scores (See Figure 2), and both groups maintained approximately the same values until the sixth month after NIBS, when there was a slight tendency for the total score in ABC, ADI-R, and ATEC to increase, but their punctuation remained lower than the observed values before treatment (See Figure 2). According to their parents' opinion, stereotypes intensity and variety increased in frequency and number in many patients around the sixth month, but in clinical scales there were not any significant changes in specific domains. Group 1 had lower values for all the scales one month after NIBS (solid lines); the same happened with G2 patients running in parallel, but only since the fourth month and ahead of follow-up (also one month after NIBS; dashed lines). BODY.3. RESULTS.3.3. EEG-BASED BRAIN FUNCTIONAL CONNECTIVITY ANALYSIS: An increase in brain functional connectivity was observed after NIBS for patients who received either tDCS or rTMS, especially for higher frequencies: α, β, and γ bands showed the greatest increase, with a large increment in the number of 10/20 system points functionally related. The analysis of the effect in functional connectivity showed that the group that received rTMS accounted for the most significant changes when initial and final values were compared (see Figure 3). BODY.3. RESULTS.3.4. ERP ANALYSIS: Recordings were possible in only six patients in the initial evaluation; thus, after the intervention, only activity in those patients who collaborated during the task was recorded. All patients had a normal auditory brainstem response according to the normative laboratory data, but one patient had no ERP response. The P300 component showed a scalp distribution in the frontal and central regions, maximal in Fz. There was a shortening of P300 latency after treatment, with statistically significant differences with respect to the basal response (Wilcoxon matched pairs test, p = 0.043). P300 amplitude did not show statistically significant differences after treatment, even though the average value in the group was higher after the intervention (see Figure 4). The prolonged latency and low amplitude may be in agreement with the abnormal connectivity of the frontal lobes seen in ASD subjects [25], and engaged with attention circuits. The improvement of these parameters after NIBS might be a consequence of the improvement in functional connectivity and probably correlated with positive clinical changes in attentiveness, concentration, and speed of answering questions from a parent. BODY.4. DISCUSSION: Our results reproduce similar neuromodulatory effects to those reported previously by two other research groups who used the same target (L-DLPFC), applying either rTMS or tDCS [9,11,26]. Casanova et al. pioneered the use of therapeutic NIBS in autism and described the effects of one weekly low-frequency rTMS (1 Hz) during 12 weeks in 25 ASD children and adolescents. In that study they reported a significant reduction in repetitive and restricted behavior patterns and irritability, but no changes in social awareness or hyperactivity. They also described an increase in the amount of gamma activity in the EEG, shortening of the P300 component of ERP, and an increase of amplitude in the group receiving the intervention [9]. They used 150 TMS pulses per session, one session per week, and 12 sessions in total. In our study, the number of stimuli per session and the total number of sessions were considerably higher; this is in correspondence with recommended practical aspects for conventional use of NIBS in other diseases such as depression, obsessive compulsive disorder, chronic pain, etc., in order to obtain a sustained effect [27,28]. This could likely explain the significant clinical improvement in our group, not only in the abovementioned domains, but also in social aspects and communication. We also were able to establish that NIBS' effects persisted into the sixth month after completion of treatment, a result that, as far as we know, has never been reported by any other group; this is a very important finding because it would probably indicate that patients would need a new treatment cycle (e.g., a second cycle of NIBS) six months after the previous intervention to maintain clinical improvement. Of course, it would also be interesting to know how the behavior of our patients will develop over the next six months and the forthcoming years (long-term outcome). Another interesting double-blind and randomized trial in adult ASD patients was published by Enticott et al. using the dorsomedial prefrontal cortex as the target, and stimulating with the HAUT coil, which makes it possible to reach deep structures in the brain [12]. They applied 10 daily sessions of 5 Hz, 1500 pulses at resting motor threshold intensity. The clinical response was evaluated immediately after the intervention and one month later, and they reported a significant reduction in social relating symptoms (relative to sham participants) for both post-treatment assessments. Those in the active condition also showed a reduction in self-oriented anxiety during difficult and emotional social situations from pre-treatment to one-month follow-up. They suggested the need to use extended protocols such as those used to treat depression, and we agree that it is essential to achieving lasting effects for both NIBS methods, rTMS or tDCS [28]. D'Urso et al. published the results of an open pilot study using cathodic tDCS over the L-DLPFC in 10 young adults (18–25 years old) with 10 daily sessions of 1.5 mA, with evaluation two weeks after the intervention. They reported an improvement in autistic behavior of 26.7% according to the change observed in the clinical scale they applied; only two patients did not change their initial scale values, while the rest of the group improved in their symptoms [11]. There are two main differences in the tDCS protocol we applied in younger children (5–10 years old): first, again we used a higher number of sessions and second, the stimulation intensity in our case was 1 mA. NIBS in children seems to be as safe as in the adult population, so the same safety guidelines are indicated for its use in any population [29,30]. It is important to mention that in the case of tDCS, there is not enough evidence about the safety of 2 mA in young people (<18 years old), and most research has been done using 1 mA; however, there are also a few papers describing the absence of adverse effects with the use of 2 mA in pediatric patients [13]. In another interesting article, Amatachaya et al. described the effects of anodal tDCS over L-DLPF, with improvement in autistic behavior in 20 children with ASD. They designed a randomized, double-blind crossover and sham-controlled trial, applying 1 mA anodal tDCS over five consecutive days, and a washout period of four weeks This result reactivates the essential research question as to whether the clinical, biochemical, and physiological effects are polarity-dependent or not [10,31,32,33,34,35]. There are results from clinical trials with other diseases and physiological studies in which the opposite effects of cathodic and anodic tDCS have been well documented [35,36]. Our results using cathodic tDCS or low-frequency rTMS over the L-DLPFC share the same theoretical basis developed by Casanova et al. and D'Urso et al., though we studied a low sample number (24 patients), and by design we employed an open trial without the use of placebo stimulation. Nevertheless, the results are reproducible in G1 and G2, with patients being their own controls from the start of the intervention. We also had the opportunity to compare the effects of low-frequency rTMS and cathodic tDCS at the same time, but in ASD patients of different ages and with our experimental design we demonstrated that there are no differences, at least not in their clinical effects. Certainly, there was an important effect achieved by increasing functional connectivity with the use of both the stimulating methods, rTMS and tDCS, with a clear superiority of rTMS-induced changes. Of course, more research is needed to draw well-documented conclusions. Other authors have described improvements in particular aspects of adult ASD patients such as working memory and better syntax acquisition following single-dose anodal tDCS over the left and right dorsolateral prefrontal cortex (first case) or exclusively over the left dorsolateral prefrontal cortex [13,37] There were also positive changes in P300 ERP, but due to the low number of subjects in whom it was possible to carry out the passive oddball paradigm, we cannot make any comparison between the two different stimulating methods. Unfortunately, sample sizes for EEG-based connectivity analysis and ERP paradigm could not include all the patients, because, in close relationship with the complexity of the used method, when we carried out their initial electrophysiological evaluation (before intervention), not all recordings had a high enough quality for analysis. BODY.5. CONCLUSIONS: Twenty sessions of NIBS over L-DLPFC improves autistic symptoms in ASD children, with a lasting effect of six months.

TITLE: Association of fatigue and depression with circulating levels of proinflammatory cytokines and epidermal growth factor receptor ligands: a correlative study of a placebo-controlled fatigue trial ABSTRACT.CONTEXT: The biology of fatigue and depression in cancer patients is poorly understood. Hypotheses regarding cytokines and growth factors related to sickness behavior and disruption of circadian signaling have been proposed. ABSTRACT.OBJECTIVES: We prospectively examined proinflammatory cytokines (e.g., sickness behavior model) and epidermal growth factor receptor (EGFR) ligands (e.g., circadian disruption model) in the serum of cancer patients enrolled in a clinical trial testing levocarnitine for fatigue. ABSTRACT.METHODS: Serum samples were collected at baseline and week 4. Cytokine/growth factor analyses were performed with a Luminex analyzer. The Brief Fatigue Index and the Center for Epidemiologic Studies Depression Index were used to measure fatigue and depression severity. The association between cytokine and symptoms was examined using logistic models. ABSTRACT.RESULTS: Among 101 analyzable patients, all ten cytokines/growth factors examined were highly elevated at baseline and all significantly decreased at week 4 (p<0.001) regardless of treatment intervention. At baseline, the odds of severe fatigue significantly increased for patients with higher level of interleukin-1 receptor antagonist (IL-1Ra), whereas patients with higher levels of IL-1Ra, tumor necrosis factor-α, interleukin (IL)-6, IL-8, interferon-γ, transforming growth factor α, and vascular endothelial growth factor had higher odds of severe depression. At week 4, fatigue (p=0.023) and depression (p=0.007) responders had less decrease in IL-1 level than the corresponding non-responders. ABSTRACT.CONCLUSION: In this correlative analysis of a fatigue clinical trial, levels of fatigue were significantly associated with levels of IL-1 and IL-1Ra. Circadian-signaling pathways related to EGFR signaling were correlated with depression as were other cytokines. A major placebo effect was associated with a global decrease in cytokine and growth factors. These data provide further basis for testing hypotheses regarding the mechanisms of fatigue and depression in cancer patients. BODY.INTRODUCTION: Fatigue is the most commonly reported symptom in cancer patients. Clinical factors that may impact the development of fatigue, such as disease progression, depression, poor nutritional status, and treatment, are often considered. The underlying molecular mechanism of fatigue, however, is still poorly understood. Biological factors that have been examined in recent years include cytokine dysregulation, hypothalamic–pituitary–adrenal axis dysfunction, 5 hydroxy tryptophan neurotransmitter dysregulation, circadian rhythm disruption, alterations of muscle metabolism, vagal afferent activation, and genomic factors.1 Two "cytokine" hypotheses regarding the production of fatigue and related symptoms in cancer patients are the focus in this report. The first is based on the "sickness behavior model"2 that is thought to be related mechanistically to elevation of serum and brain levels of proinflammatory cytokines of the interleukin (IL) family (e.g., IL-1, IL-1Ra, and IL-6).3–9 Activation of this cytokine signaling in the peripheral and central nervous system by cancer or cancer treatment has been shown to lead to malaise, poor appetite, and social withdrawal, which are believed to underlie fatigue, cognitive disturbances, and depression.10,11 The second hypothesis is that symptoms could be due to the inhibition of circadian-signaling pathways. This hypothesis is based on extensive laboratory data12 showing inhibition of downstream hypothalamic signaling from the master clock in the suprachiasmic nucleus and corroborated by clinical observations.13 The inhibition of these hypothalamic signaling pathways can produce fatigue, suppress feeding activity, and cause malaise. Elevation of circulating levels of ligands of the epidermal growth factor receptor (EGFR), such as epidermal growth factor (EGF), transforming growth factor α (TGF-α), and neuregulin,14,15 can act to inhibit neural signaling that drives normal behaviors, in laboratory studies. The patients reported here were enrolled on a treatment intervention study conducted by The Eastern Cooperative Oncology Group (ECOG) E4Z02: a phase III, randomized, double-blinded, placebo-controlled study to determine the effectiveness of levocarnitine supplementation in the management of moderate-to-severe fatigue in cancer patients. The mechanism for this intervention and the preliminary laboratory and clinical data supporting this have been described previously.16 The final analysis of the primary endpoint showed no significant difference in fatigue between levocarnitine and placebo treatments.16 Shortly after the E4Z02 study opened, this correlative laboratory study of ten serum cytokines was initiated based on the hypotheses mentioned above. The specific objectives of the correlative study are: 1) to measure serum levels of proinflammatory cytokines and growth factors (hereafter referred to as cytokines), and 2) to correlate cytokines with fatigue and depression. BODY.PATIENTS AND METHODS.PATIENTS AND TREATMENT: xmlns:ns0="http://www.w3.org/1999/xlink">A subset of the E4Z02 patients was included in the current report (Figure 1). All patients were enrolled to E4Z02 through ECOG-affiliated institutions and were recruited when they checked in for their clinical appointment. Eligible patients were required to have a confirmed invasive malignant disorder (at any stage of any site and at any point in their cancer treatment) and have moderate-to-severe fatigue (defined as a score of ≥2 on a Likert-type 0–4 rating scale from the Functional Assessment of Chronic Illness Therapy (FACIT-F; http://www.facit.org/) question: "I feel fatigued") recorded as an outpatient within 4 weeks prior to registration. More details about patient eligibility criteria could be found in Cruciani et al.17 The protocol was approved by the Eastern Cooperative Oncology Group (ECOG) national review board at each participating institution. After providing written informed consent, patients were randomized in a 1:1 ratio to receive 2000 mg/day (two 1000 mg doses) of either levocarnitine or placebo (both in liquid form) for 4 weeks. For weeks 5–8 (extension phase), all patients received 2000 mg/day of open-label levocarnitine.16 BODY.PATIENTS AND METHODS.ASSESSMENT OF SYMPTOMS: All symptom assessments were completed at baseline (prior to the initiation of levocarnitine or placebo) and week 4 (after starting protocol therapy). Severity of fatigue was assessed using the Brief Fatigue Inventory (BFI).17 The total fatigue score (range: 0–10) was the average score for all nine items in BFI, with a higher score indicating worse fatigue. Severe fatigue, however, is defined as scores ≥7 for the "worst" fatigue level during past 24 hours item due to "its conceptual simplicity and close relation to function interference".17 Severity of depression was assessed with Center for Epidemiologic Studies Depression Scale (CES-D).18,19 The total depression score (range: 0–60) was the sum of the scores for all 20 items, with a higher score indicating worse depression. A total score of ≥16 was considered a potentially significant level of depression,18,19 which was called severe depression in the study. Compared with baseline, a ≥50% improvement at 4 weeks of the BFI and CES-D total scores was considered a symptom response in the present analysis.20 As a sensitivity analysis, symptom response was also defined as ≥4-point decrease for BFI total score and ≥10-point decline for CES-D total score at week 4 assessment. In the sensitivity analysis, symptom response was only examined in patients with baseline score of no less than the above-specified cutoff points. The above cutoff points were chosen because we believed those levels of improvement in fatigue and depression could be considered clinically meaningful. BODY.PATIENTS AND METHODS.SERUM COLLECTION AND CYTOKINE ELISA ANALYSIS: After activation of the amendment incorporating this cytokine correlative study into E4Z02 trial, serum samples were collected from consenting patients at baseline (prior to the initiation of protocol therapy) and week 4 (prior to first treatment of the extension phase). The baseline blood draw was done between 6 and 10 am (8 am optimal) before receiving cancer therapy if applicable. The week 4 blood draw was collected at the same time of day as the baseline blood. After the blood was drawn, the blood was centrifuged at ~3,500×g at 4°C for 10 minutes to separate the serum. The serum was then drawn into a sterile syringe (or a transfer pipette) and then evenly dispensed (aliquot) into labeled cryotubes in 2-mL aliquots. The vials were capped securely and the serum was frozen at −70°C or colder at the local institutions and shipped to the central ECOG Pathology Coordinating Office repository after collection of both baseline and week 4 evaluation samples. After all the samples were collated upon completion of the clinical study, the ECOG Pathology Coordinating Office shipped these samples to investigators at the University of Virginia, where all cytokine analyses were conducted. The ten cytokines assayed were IL-1, IL-1β, IL-6, IL-8, IL-1Ra, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), EGF, TGF-α, and vascular endothelial growth factor (VEGF). Cytokines IL-1β, IL-6, and TNF-α were tested via the ELISA kits from DRG International, Inc (Springfield, NJ, USA). TGF-α and EGF were tested using ELISA kit QIA61 from Oncogene Research Products (Cambridge, MA, USA), and VEGF was tested using ELISA kit DVE00 from R&D Diagnostics (Minneapolis, MN, USA). The cytokine analysis was performed with a Luminex analyzer on first thawed samples using up to 500 μL for duplicate assays. Detection was achieved through a bead-based antibody–antigen sandwich method. Briefly, samples were incubated with color-coded beads that were precoated with analyte-specific capture antibodies for the molecule of interest. Expression levels were determined following incubation with a biotinylated detection antibody and streptavidin-conjugated phycoerythrin (PE). Using a Luminex® analyzer, independent lasers determined the color of each bead and the magnitude of the PE-derived signal, which was directly proportional to the levels of bound analyte. Samples were given a missing value for cytokines if the levels of cytokines were below the detection level of the kits. Samples with missing values for cytokine levels were excluded from corresponding analysis. Hence, the sample size might vary for different cytokines. The average of the duplicates was used for each cytokine in all analyses. BODY.PATIENTS AND METHODS.STATISTICAL ANALYSIS: The target accrual for the correlative serum study was 160 patients to give an 80% power to detect a 0.5 standard deviation (SD) difference in mean serum levels of any of the cytokines between patients with severe and non-severe symptoms, using Wilcoxon rank sum test with a 0.05 two-sided type I error. Wilcoxon signed-rank test and rank sum test were used to examine the difference between groups for continuous variables. Fisher's exact test was used to test the association between categorical/binary variables. Multivariable linear and logistic models and generalized estimating equation (GEE) models were used for regression analysis. No adjustment was made for multiple comparisons in this exploratory analysis. All p values were two-sided and a p value of 0.05 was considered statistically significant. STATA 11.2 (StataCorp., College Station, TX, USA) was used for all analyses. BODY.RESULTS.PATIENT CHARACTERISTICS: A total of 376 patients were enrolled to E4Z02 between November 2005 and January 2007, and 172 of them were enrolled after the activation of the correlative study in September 2006. Serum samples were available for 101 patients at baseline and 4 weeks after initiation of protocol therapy; 47 on levocarnitine, and 54 on placebo. Figure 1 displays the study CONSORT diagram. No significant difference in baseline patient characteristics was found except for disproportionately lower overall metastatic disease rate among the 101 patients compared with the remaining 275 patients without cytokine assessment (data not shown). The baseline symptom levels were similar as well (Figure 2A, C). Of the 101 patients with cytokine data, the patient characteristics were well balanced between the levocarnitine and placebo arms except for a lower proportion of males in the levocarnitine arm (Table 1). Hence, we analyzed the data regarding the symptoms correlated with cytokines on the collapsed population of 101 patients. BODY.RESULTS.FATIGUE AND DEPRESSION AT BASELINE AND WEEK 4 ASSESSMENTS: Table 2 shows a significant decrease in fatigue and depression levels between baseline and week 4. Of the 101 patients, 64.4% had severe fatigue at baseline and 54.0% reported severe fatigue at 4-week assessment. The mean percent change of BFI total score was −17.4%, and 16.2% (n=16) of patients had a ≥50% improvement in BFI total scores (fatigue responders). For depression, 44.6% and 36.0% of patients reported severe depression at baseline and week-4 assessments, respectively. The mean percent change of the CES-D total scores was −5.4%, and 24.2% (n=24) of patients had a ≥50% improvement in CES-D total scores (i.e., they were depression responders). As illustrated in Figure 2B, D, symptoms improved significantly for all patients independent of treatment arm. BODY.RESULTS.CYTOKINES AT BASELINE AND WEEK 4 ASSESSMENTS: The mean serum levels for all cytokines are displayed in Table 2. All cytokines were elevated at baseline compared with the normal levels from the literature. Multivariable linear regression analyses show that patients with current radiotherapy had a higher level of IL-1β (p=0.002), IFN-γ (p=0.026), and TGF-α (p=0.03) at baseline, after adjusting for other covariates. Patients with metastatic disease had a higher level of TNF-α (p=0.047) and IL-8 (p=0.044). Male patients had a higher level of IL-1β (p=0.028) and IL-1Ra (p=0.023; data not shown). Overall, the mean serum levels significantly decreased between baseline and week 4 for all cytokines (p<0.001 for all cytokines, Table 2). The GEE analyses showed that the most significant cytokine decreases over time were on the placebo arm and in patients currently receiving irradiation (data not shown). BODY.RESULTS.ASSOCIATION BETWEEN CYTOKINES AND SYMPTOMS AT BASELINE: There was no statistically significant difference in cytokine levels between symptom groups (severe vs. non-severe fatigue and severe vs. non-severe depression) at baseline based on the Wilcoxon rank sum test (p>0.05 for all cytokines, data not shown). However, patients with higher levels of IFN-γ (odds ratio [OR]=1.45, p=0.013), IL-1Ra (OR=1.62, p=0.022), IL-6 (OR=1.45, p=0.015), IL-8 (OR=1.85, p=0.003), TNF-α (OR=1.79, p=0.004), TGF-α (OR=1.57, p=0.019), and VEGF (OR=1.75, p=0.027) had statistically significantly higher odds of having severe depression at baseline, after adjusting for available confounding variables in the multivariable logistic models (Table 3). Patients with a higher level of IL-1Ra also had increased odds of having severe fatigue at baseline (OR=1.57, p=0.033; Table 3). BODY.RESULTS.ASSOCIATION BETWEEN CYTOKINE CHANGE AND SYMPTOM RESPONSE: Table 4 displays the association between cytokine change and symptom response. Between the week 4 and baseline assessments, both fatigue (p=0.023) and depression (p=0.007) responders had significantly less decrease in IL-1 level compared with the corresponding non-responders. Furthermore, depression responders had significantly less decrease in IFN-γ (p=0.005) and VEGF (p=0.045) levels as well. When symptom response was evaluated by absolute score change, IL-1 was still significantly associated with symptom response for both fatigue and depression, and IFN-γ and VEGF were no longer statistically significant for depression (data not shown). BODY.RESULTS.SENSITIVITY ANALYSIS FOR ASSOCIATION BETWEEN CYTOKINES AND SYMPTOMS: A sensitivity analysis was conducted where the lowest detection level was imputed for samples with levels of cytokines below that. The results about association between cytokines and symptoms were similar in the subjects where samples were given missing values in the same situation (data not shown). BODY.DISCUSSION: This prospective study of a heterogeneous group of patients with invasive malignancy and moderate-to-severe fatigue shows that all cytokines are elevated at baseline compared with the normal levels reported in other studies.21,22 The very high levels found here may be related to factors known to elevate cytokine levels like stress, chronic illness, irradiation, and infection. Certain patient demographic and disease characteristics were found to be associated with higher levels of cytokines in our study, such as metastatic disease, current radiotherapy, and male sex. Performance status per se is not associated with cytokine levels in our study. Consistent with our study hypothesis, higher levels of cytokines appear to be associated with a greater symptom burden (Table 3). At the baseline assessment, a multivariable analysis shows that a high level of IL-1Ra is associated with severe fatigue. This observation is consistent with a study of fatigued breast cancer and prostate cancer survivors.23 A meta-analysis of over 1000 cancer patients also found that the circulating levels of IL-1Ra and IL-6 are associated with fatigue.24 In our multivariable analysis for depression, seven (IL-1Ra, TNF-α, IL-6, IL-8, IFN-γ, TGF-α, and VEGF) of the ten cytokines are significantly associated with severe depression. This observation about IL-6 is consistent with previous studies.6,25 Cancer-related fatigue is extensively studied because it is prevalent and associated with significant morbidity, functional impairment, and loss of quality of life.26 Several studies have been conducted to examine whether targeted interventions based on molecular mechanisms could help alleviate fatigue. For example, a study of the prospective use of dexamethasone for fatigue in advanced cancer patients showed modest benefit at 2 weeks.27 Another recent trial using the anti-IL-6 antibody ALD 518 has shown some promising results and indicates that the IL-6 pathway disruption may be a promising avenue to pursue.28 The multidimensional causes of cancer fatigue production, in particular, the possible relationship with disruption of the biologic timing mechanism by ligands of the EGFR family, is another potential therapeutic intervention. Supporting this notion are the results with the tyrosine kinase inhibitor, gefitinib, that showed early reversal of multiple symptoms that are hard to explain solely on the basis of tumor response.29 Furthermore, in a small pilot trial, we found that the use of the tyrosine kinase inhibitor did reverse objective measurements of the circadian system with normalization of wrist actigraphy measurements, indicating fatigue in a cohort of patients with metastatic non-small cell lung cancer.30 Future studies with anti-EGFR agents should explore the potential for improvement of symptoms as well as antitumor activity in the quest for better understanding of the interventional potential of this class of agents. In the parent E4Z02 trial16 and in our correlative cytokine study reported here, and in a separately published randomized fatigue intervention trial,31 improvement in symptoms occurred independent of the therapeutic intervention and represents a placebo effect. We show here that fatigue improvement is associated with a decline of the IL-1/IL-1Ra pathway in this population that experience the placebo effect (Table 2), thus contributing to the understanding of fatigue within the emerging knowledge of the importance of neural-chemical pathways in producing placebo effects per se.32 Although the precise neurochemical mechanism is unknown, the observed placebo effects can be important confounding factors in clinical symptom research. One approach to account for this would be to assess patient's expectations, a dimension that has been shown to influence treatment intervention in studies concerning pain and inflammation.33 Another way could examine the actual language used by health workers (e.g., certified research assistants and doctors) enrolling patients onto clinical trials that could provide an elevated sense of anticipation that has been shown to influence outcomes positively or negatively (placebo or nocebo, respectively).32 There are some important limitations for this correlative study. First, only one follow-up was made, at week 4. Multiple evaluations of the cytokine levels at specific times as well as follow-up extended beyond 1 month might be important, given the known circadian variation in these cytokine levels as well as changing trajectory of fatigue over time. Second, various additional variables were not collected (e.g., duration of fatigue before study entry, body mass index, menopausal status of female subjects, sleep problems, concomitant use of sleep aid, nutrition and oxygen status, the type and the timing of chemotherapeutic agents used, hormone therapy, and immunotherapy). Some of these additional variables might mediate the association between cytokines and fatigue. Third, some details about the blood draw (e.g., whether it is collected before taking the study drug intervention) are not explicitly specified in the protocol, which might have an impact on the study results. Fourth, we did not adjust these exploratory analyses for multiple comparisons. Finally, the analyses are underpowered due to the small sample size. Overall, these correlative study data should be considered as exploratory and hypothesis generating. In summary, this correlative study of a placebo-controlled trial involving a drug intervention to treat fatigue indicates that there are elevations in proinflammatory cytokines (IL-1 and IL-1Ra) and ligands of the EGFR family that are correlated with fatigue and depression in cancer patients. Correlative studies are an important component of placebo-controlled symptom interventions, providing biological insights and leads that enhance the value proposition of this research even when the investigational treatments lack efficacy.

TITLE: Benefits of different postoperative treatments in patients undergoing knee arthroscopic debridement ABSTRACT.PURPOSE: To assess the effectiveness of viscosupplementation or platelet-rich plasma (PRP), compared to standard care, for pain relief after knee arthroscopic debridement in patients with meniscal pathology and osteoarthritis (OA), under normal clinical practice conditions. ABSTRACT.PATIENTS AND METHODS: We conducted a prospective, randomized, evaluator-blind, pilot study. After arthroscopy, patients were randomized to receive 1) five injections of HA1 (Suprahyal®/Adant®); 2) four injections of HA2 (Orthovisc®); 3) three injections of HA3 (Synvisc®); 4) a single injection of PRP (GPSTM II); or 5) standard care (control). Patients were followed up for 18 months. Clinical outcomes were evaluated using the Western Ontario and McMaster Universities Arthritis Index (WOMAC) at 3, 6, 12, and 18 months. Minimally Clinical Important Improvement (MCII), as relative improvement ≥20 for pain and function, was also calculated. ABSTRACT.RESULTS: Fifty patients were included. At early follow-up (3 months), total WOMAC scores improved in all groups compared to baseline with reductions of 44.79% (HA1), 24.02% (HA2), 40.38% (HA3), 39.77% (PRP), and 27.64% (control) (p=0.002 HA1 compared to HA2). At 18 months, the higher improvement in total WOMAC was in HA1 with a 65.20% reduction, followed by PRP (55.01%), HA3 (49.57%), and HA2 (29.82%), whereas the control group had a 14.55% increase over baseline (p=0.001 control compared to HA1 and HA3). The percentage of patients achieving the MCII for both pain and function at 18 months was 100% (HA1), 80% (HA3), 60% (HA2), and 60% (PRP), whereas, in the control group, all patients returned to pre-arthroscopy levels. There were no adverse events attributable to surgery or to intraarticular administration. ABSTRACT.CONCLUSION: Viscosupplementation following arthroscopy is more effective than PRP in adequately selected patients with meniscal lesions occurring concomitantly with OA. Further controlled studies with a larger sample size and/or alternative regimens would be of interest for the scientific community. BODY.INTRODUCTION: Osteoarthritis (OA) is the most common of all joint diseases and exacts a heavy economic toll due to its high prevalence in the general population and potential for causing progressive disability.1 The etiology is multifactorial, including a variety of risk factors (aging, genetics, trauma, malalignment, and obesity) that interact to cause this disorder.2 The primary goal of treatment is the alleviation of pain, leading to an improvement in joint function and quality of life. Treatment options for knee OA include conservative measures, pharmacological treatments, intraarticular (IA) injections, and surgical intervention.3 Viscosupplementation is a well-established treatment option in OA. Hyaluronic acid (HA) exerts a mechanical effect providing lubrication of the joint, protecting against loads and impacts, and restoring the rheological properties of the synovial fluid.4 HA also has a pharmacological effect by interacting with mediators of inflammation, inhibiting nociceptors of pain, stimulating chondrocyte growth, synthesis of extracellular matrix protein, and reduction of apoptosis in osteoarthritic cartilage.5–8 HA products that are available differ in many characteristics, including origin (animal vs biofermentation), molecular weight (MW), structure (linear, crosslinked, and mix of both), volume of injection, and posology. Clinical investigations with these products have been mainly focused on the potential differences attributable to MW. Arbitrarily, different HA products have been divided into three MW categories: low (500–730 kDa), intermediate (800–2,000 kDa), and high MW (2,000–6,000 kDa) including crosslinked formulations of HA.9 The therapeutic use of platelet-rich plasma (PRP) is based on the use of the patient's own platelets as carrier of growth factors and other proteins (eg, fibronectin and other adhesive proteins) that play an important role in cell biology. The resulting products vary in their leukocyte content, platelet concentration, subsequent activation, and, at this moment, it is not defined what should be the ideal concentration of platelets that should reach the preparations as well as the dosage regimen.10 Although it seems PRP may improve the symptomatology, one part of the scientific community considers that there still exists a lack of clarity in available evidence for this to be recommended in international guidelines.11 Knee arthroscopy has become the gold standard in the diagnosis of meniscal and ligamentous injury12 and also plays a role in their management.13 This intervention has been widely used as a treatment for OA of the knee, but recent studies14–16 with controversial design and methodology17 have questioned its usefulness. However, it should be noted that >90% of patients with symptomatic knee OA have magnetic resonance imaging findings compatible with meniscal pathology.18 Therefore, arthroscopic surgery would be indicated in those patients with meniscal injuries, loose bodies, osteochondral lesions, or synovial pathology, with a concomitant diagnosis of knee OA playing an essential role in the adequate selection of patients who can benefit from this technique.19 Arthroscopy has decreased morbidity as compared with that in open procedures, but it has not eliminated pain completely. The causative factors behind this persistent pain are unknown, although it is known that most of the IA structures of the knee, including the synovial tissue, the anterior fat pad, and the joint capsule, have free nerve endings that are capable of sensing painful stimuli and producing severe pain.20,21 Moreover, it has been postulated that the severity and extent of OA, the degree of debridement undertaken, and the skill of the surgeon could be factors involved.22 Considering the abovementioned aspects, this study was planned as a pilot to assess the effectiveness of different treatments such as viscosupplementation or PRP as compared to standard care for pain relief after knee arthroscopic debridement in patients with meniscal pathology and OA, under normal clinical practice conditions. BODY.PATIENTS AND METHODS.ETHICS: The study was conducted in accordance with the Good Clinical Practice guidelines and in compliance with the principles of the Declaration of Helsinki. The study protocol was approved by the local Ethics Review Board from Hospital Español de México, and all patients gave written informed consent to participate in the study. BODY.PATIENTS AND METHODS.DESIGN: This was a prospective, randomized, evaluator-blind, pilot study with four parallel arms and a control group. Eligible patients were informed about study purpose and design. Demographic characteristics and medical history of the participants were recorded, and laboratory tests were done. X-rays of both knees were performed using anteroposterior projection with support, lateral with 30° flexion, and Merchant (45°) views; bipodalic mechanical axis digitalization as well as magnetic resonance imaging of the affected knee were also done. Arthroscopies were all carried out by a single orthopedic/arthroscopic surgeon. All patients were given three doses of antibiotic (cephalothin 1 g) as prophylaxis. Postsurgical pain treatment comprised oral acetaminophen 1 g/8 h and celecoxib 200 mg/12 h for 10 days. After surgery, patients were randomized to receive: 1) five IA injections of HA1 (Suprahyal®/Adant®: biofermentation, non-crosslinked, 600–1,200 kDa, 25 mg/syringe); 2) four IA injections of HA2 (Orthovisc®: biofermentation, non-crosslinked, 1,700–2,900 kDa, 30 mg/syringe); 3) three IA injections of HA3 (Synvisc®: chicken combs, crosslinked, 7,000 kDa, 16 mg/syringe); 4) a single IA injection of PRP (GPS® II); and 5) standard care. The HAs used were considered of interest as they have different characteristics, 23, 24 to study whether these differences could have any impact on the efficacy/safety results. The number of doses specified are those authorized for each product.23 The GPS® II is a closed system that enables PRP to be collected while avoiding the risk of contamination and makes possible to concentrate the autologous platelets for reinjection locally.25 Eligible patients were randomized 1:1 using a computer-generated list of random numbers. The random sequence was created using freely accessible tools available at http://www.randomization.com, which uses the pseudo-random number generator of Wichmann and Hill, 26 modified by McLeod.27 PRP was administered immediately after arthroscopy and viscosupplements were given once a week, starting at 21 days post-arthroscopy. Rescue medication comprised acetaminophen 1 g/8 h and celecoxib 200 mg/12 h. The administration of IA treatments was conducted under aseptic conditions, inserting the needle into the patellofemoral joint space by a superolateral approach, with the patients in a supine position. In case effusion was present, it was removed before each injection. Follow-up was conducted by five orthopedic surgeons, each one assigned to a specific group and blinded to the treatment administered. Visits were scheduled at 3, 6, 12, and 18 months after arthroscopy. All patients were prescribed the same protocol of rehabilitation to be followed at the patient's convenience. The rehabilitation protocol started progressively, starting with isometric exercises and muscle stretching from Week 1 post-arthroscopy, adding weight exercises from Week 3, cardiovascular training from Week 7, and high-impact exercises from 3 months onward. BODY.PATIENTS AND METHODS.PATIENT SELECTION CRITERIA: Eligible patients were men and women, aged 40–85, with body mass index (BMI) <35 kg/m2, and indications for knee arthroscopic surgery due to symptomatic meniscal rupture Mink et al28 grade III confirmed by magnetic resonance imaging, radiographic OA grades I–II according to Kellgren and Lawrence (KL),29 meniscal preservation >60%, ligament integrity, and ability and willingness to provide informed consent for study participation. Main exclusion criteria were infectious conditions, use of anticoagulants, history of trauma (dislocation or fracture), inflammatory arthritis, microcrystalline arthropathies, history of septic arthritis, ligament injury, nonspecific synovitis, angular deformity >10°, chondral lesions G-IV Outerbridge (>1 cm2),30 neoplasms, or allergy to any of the components of the products under study. BODY.PATIENTS AND METHODS.EVALUATION OF EFFICACY: Efficacy was evaluated using the Western Ontario McMaster University Osteoarthritis Index (WOMAC) total and subscales of pain, stiffness, and function using a five-point (0–4) Likert scale. Scores at the end of follow-up (18 months) were compared to baseline and the differences between treatment groups were analyzed. Secondary outcomes were efficacy rates at the different follow-up visits. The WOMAC questionnaire used was the Spanish translated version from Batlle-Gualda et al,31 validated by Escobar et al.32 The number of patients achieving the Minimal Clinically Important Improvement (MCII) in each treatment group was also calculated. The MCII is the smallest change in measurement that signifies an important improvement in a patient's symptom.33 According to Tubach et al,34 a 15 out of 100 for absolute improvement and 20% for relative improvement could be used as cutoffs for MCII estimation. Taking into account that, in our study, a Likert scale was used, the 20% of relative improvement cutoff was applied.33 BODY.PATIENTS AND METHODS.EVALUATION OF SAFETY: The incidence and type of adverse events was recorded throughout the study. BODY.STATISTICS: Categorical variables were summarized by their number and relative frequencies. For continuous variables following a normal distribution (Kolmogorov–Smirnov test), mean, standard deviation, and maximum and minimum values were used. Non-normally distributed variables were summarized with their median, interquartile range, minimum, and maximum. Changes in WOMAC total and subscales at 3, 6, 12, and 18 months were expressed as raw scores and as percentage of change from the baseline value. Comparisons between groups were done using chi-square test or Fisher's Exact test for categorical variables when necessary. Continuous data were analyzed using Mann–Whitney test. The study was designed as a pilot evaluation; therefore, real sample-size estimation was not done. The main population for analysis was the per protocol (PP) population, comprising patients who completed the 18 months follow-up according to the study protocol. Safety analysis was conducted in all patients who received at least one IA injection. Data were analyzed using SPSS V14 (SPSS Inc, Chicago Il). All tests were two-sided, and the statistical significance was set at 0.05. BODY.RESULTS.DISPOSITION OF PATIENTS AND DEMOGRAPHICS: Patients were recruited from March 2013 to October 2014. Ninety patients were initially screened, of whom 40 were excluded due to age (n=4), history of trauma or ligament involvement (n=5), overweight (n=6), meniscal injury > 40% (n=4), chondral lesions G-IV (Outerbridge >1 cm2; n=5), eventual total knee arthroplasty (TKA) (n=7), lost to follow-up (n=8), or death unrelated to the study (n=1), leaving a total of 50 patients who participated in the study and completed all procedures as scheduled (Figure 1). Among study subjects, 52% were male and the mean age was 64.4 years, with statistically significant differences between HA2 and HA3 (p=0.007). Mean BMI was 27.8 kg/m2 without differences among groups (p=0.693), despite HA2 comprising more obese patients (Table 1). Near all patients in the HA3 group had mild OA (KL grade I), with the differences significant versus HA2 (p=0.001) and control (p=0.002). The groups were homogeneous with respect to WOMAC total values (p=0.062) as well as pain (p=0.234), stiffness (p=0. 383), and function (p=0.089) subscales (Table 1). There were no differences among groups in arthroscopic lesions (Table 2). The following surgical procedures were conducted: partial meniscectomy of no more than 40% (35 patients), synovectomy and resection of Hoffa's fat pad (five patients), and debridement and stabilization of the chondral lesion (16 patients). More than one procedure in the same patient was possible. BODY.EFFICACY: At early follow-up (3 months), total WOMAC scores improved as compared to baseline, in all groups with reductions of 44.79% (HA1), 24.0% (HA2), 40.38% (HA3), 39.77% (PRP), and 27.64% (control), (p=0.002 HA1 compared to HA2). At 18 months, a higher improvement in total WOMAC was observed in HA1 with a 65.20% reduction, followed by PRP (55.01%), HA3 (49.57%), and HA2 (29.82%), whereas the control group had a 14.5% increase over baseline despite the use of rescue medication and rehabilitation, with the differences statistically significant with respect to HA1 and HA3 (p=0.001; Table 3 and Figures 2A–D). At 3 months follow-up, the percentage of patients achieving the MCII for both pain and function (20% of relative change) in different groups was 80% (HA1), 20% (HA2), 40% (HA3), 30% PRP, and 30% (control). At the end of follow-up, a growing number of patients experienced a clinically significant improvement in HA1 with a 100% responders rate, followed by HA3 (80%). In the group treated with HA2 and in those treated with PRP, the results reached 60% responders, whereas in the control group, all patients returned to near pre-arthroscopy levels of pain and impairment and none of the patients reached the 20% MCII cutoff (Figure 3). BODY.SAFETY: Throughout the overall study period, patients did not experience adverse events attributable to surgery or to IA injections. BODY.DISCUSSION: Arthroscopic surgery is frequently advocated as a treatment option to relieve symptoms of painful degenerative knee conditions. The rationale is that it may improve symptom and functions, has minimal morbidity, provides a therapeutic options, and documents the stage of the disease process. The duration of effect, however, is variable.35 Adjunctive therapies such as nonsteroidal anti-inflammatory drugs and other medications, physiotherapy, and IA steroid injections may be incorporated to improve the results.36,37 The use of arthroscopic surgery in the treatment of OA has generated much discussion. Whereas some studies14,15 reported no differences with placebo arthroscopy, other studies confirm that, in patients with preoperative mechanical symptoms secondary to loose bodies, chondral flaps, and meniscal pathology, the use of arthroscopic surgery is beneficial.38,39 The key point is to offer knee arthroscopy to selected patients. Patients with symptomatic meniscal tears, few degenerative changes, normal axis, and acute mechanical complaints can benefit from this intervention, especially if the OA is mild or moderate.40–42 Favorable results have been also shown for the management of the infrapatellar fat pad impingement and Hoffa's disease.43,44 Chronic complaints, previous surgical intervention, degenerative changes in two or three compartments, significant axis deviation, ligament imbalance, or severe cartilage destruction are among the characteristics of patients who may be candidates for unsatisfactory results after arthroscopy.42 As a result, arthroscopic knee surgery is the most common type of orthopedic surgery performed in the developed world.45 However, in patients with symptomatic OA, pain can persist after arthroscopic meniscectomy and/or debridement of chondral lesions. Treatment combining arthroscopic surgery and viscosupplementation has been widely used for patients with combined intraarticular mechanical and articular surface pathology.40 Initially, rheological characteristics that confer a lubricating effect to HA were considered the primary mechanism of action in treating OA pain. Nevertheless, rather than being a mere device, HA has been shown to exert other effects such as inhibition of tissue nociceptors, inhibition of matrix metalloproteinases, free radicals scavenging, and stimulation of endogenous HA production. Due to this, viscosupplementation has been recommended as adjunctive therapy after arthroscopic surgery as a suitable way of achieving long-term stabilization of the treatment outcome.40,44,46 The application of PRP to patients with OA was developed because of the physiological roles of several bioactive proteins and growth factors expressed in platelets, which lead to tissue regeneration. Despite the use of PRPs having been extended to the treatment of several musculoskeletal injuries the last few years, we have not found published studies analyzing the efficacy of PRP as adjunctive treatment in post-arthroscopy patients. A recent meta-analysis found that at 6 months post injection, PRP and HA had similar effects with respect to pain relief and functional impairment. However, at 12 months, PRP was associated with better improvement scores.47 In our study, the indication for knee arthroscopy was symptomatic rupture of a meniscus with symptomatic osteochondral detachment in patients with grade II OA. In these patients, surgery is the only and necessary indication, and they were given additional IA treatment with hyaluronate or PRP due to the presence of OA. In the market, there are a variety of HAs and our aim has been to verify if the differences between HA products has any clinical relevance, as well as to study the effects of PRP after knee arthroscopic debridement. Procedures for clinical practice and the dosage described by the manufacturers have been used; according to the studies published, treatments with HA usually started 3 weeks after surgery.3,35,44,48,49 In absence of recommendations in the literature, PRP injection was administered immediately after arthroscopy. The HA1 product showed the best results, with a very significant improvement of the symptomatology compared to the baseline that was maintained up to 18 months post-arthroscopy (reduction of −65.20% in total WOMAC) and with 100% of the patients reaching the MCII 20% in pain and function, followed by HA3 with a reduction of −49.57 in total WOMAC and 80% of patients reaching the 20% MCII cutoff. These products differ in origin, MW, structure, and number of injections administered and, in our study, the differences between both did not reach statistical significance. However, it should be noted that 8/10 patients in the HA3 group were KL grade I, which is associated with better response data.40,41 The HA2 group showed much more discreet and erratic results, with final reduction in WOMAC at the end of the study and nearly 30% and 60% of patients complying with MCII requirements. It should be noted that, in this group, all patients had OA grade II as well as a greater number of overweight/obese patients – a characteristic that may also have influenced the results.44 A recent meta-analysis found that, at 6 months post injection, PRP and HA had similar effects with respect to pain relief and functional impairment. However, at 12 months, PRP was associated with better improvement scores.47 In our study, patients treated with PRP constitute a diverse group who seem to follow the rule of all or nothing, so that those who improve do so in a very significant way and this improvement is maintained until the end of follow-up, whereas something similar occurs with non-responder patients; thus, at the end of the study, only 60% of patients achieved the MCII threshold. Ayhan et al50 suggest that PRP therapy would be indicated for patients <60 years and with a BMI <30. In our study, the best results in the PRP group were also seen in patients with a BMI <30, as well as in those <60 years. We did not find any another relationship between the type of response and the characteristics of the patients or the arthroscopic findings. Surprisingly, the control group showed low improvement in symptomatology at early follow-up that worsened along time with return to pre-arthroscopy levels at the 12-month follow-up that required the consumption of rescue medication. One of the weaknesses of our study is the small sample size as it was designed as a pilot evaluation with the limitations that it entails when establishing comparisons; moreover, the fact that each group was evaluated by a different person may have influenced the evaluation of the results. Finally, not all patients undertook the rehabilitation protocol in the same way, as they were treated by different people and in different clinics according to the personal needs of the patient: a more strict control of this procedure would have been desirable. In our favor, the long follow-up period allowed us to assess the evolution of the results in the long term; the criteria used for patient selection was of special relevance to select those who may benefit most from the procedure, as was the presence of a control group. The design of the study also allowed us to study the behavior of three different HAs used within routine practice and to compare them with PRP as a new therapeutic approach. Viscosupplementation is an effective treatment for the treatment of knee OA. Based on our results, we consider that its use in combination with arthroscopic surgery can provide very good results in adequately selected patients with an individualized and multidisciplinary follow-up. However, due to the importance of this type of intervention, further controlled studies with a larger sample size and/or alternative posology regimens would be of interest for the scientific community.

TITLE: Cost-effectiveness of ranibizumab in treatment of diabetic macular oedema (DME) causing visual impairment: evidence from the RESTORE trial ABSTRACT.BACKGROUND/AIMS: To evaluate the cost-effectiveness of ranibizumab as either monotherapy or combined with laser therapy, compared with laser monotherapy, in the treatment of diabetic macular oedema (DME) causing visual impairment from a UK healthcare payer perspective. ABSTRACT.METHODS: A Markov model simulated long-term outcomes and costs of treating DME in one eye (BCVA ≤75 letters) based on data from the RESTORE Phase III trial. Outcomes measured in quality-adjusted life-years (QALYs) were simulated for a 15-year time horizon based on 12-month follow-up from RESTORE and published long-term data. Costs included treatment, disease monitoring, visual impairment and blindness (at 2010 price levels). ABSTRACT.RESULTS: Ranibizumab monotherapy resulted in a 0.17 QALY gain at an incremental cost of £4191 relative to laser monotherapy, yielding an incremental cost-effectiveness ratio (ICER) of £24 028. Probabilistic sensitivity analysis showed a 64% probability of being cost-effective at a threshold of £30 000 per QALY. Combined ranibizumab and laser therapy resulted in a 0.13 QALY gain at an incremental cost of £4695 relative to laser monotherapy (ICER £36 106; 42% probability of ICER <£30 000). ABSTRACT.CONCLUSIONS: Based on RESTORE 1-year follow-up data, ranibizumab monotherapy appears to be cost-effective relative to laser monotherapy, the current standard of care. Cost-effectiveness of combination therapy is less certain. Ongoing studies will further inform on disease progression and the need for additional ranibizumab treatment. BODY.INTRODUCTION: Diabetic macular oedema (DME) is the most frequent cause of vision impairment in people with diabetes and can lead to blindness if left untreated. Even when patients receive optimal treatment with the current standard of care for DME, laser photocoagulation, improvements in vision are relatively uncommon and many patients lose vision despite laser therapy.1 2 This continued vision loss is the result of structural and physiological damage to the retinal capillary bed, and from progressive and permanent damage to the macular pigment epithelium, associated with poor control of blood glucose, blood pressure and lipid levels (the three main systemic risk factors for diabetic retinopathy and DME).3 The UK prevalence of visual impairment due to DME is estimated at approximately 3% of the adult (aged 18+ years) diabetic population.4 Visual impairment places a substantial socio-economic burden on patients, their caregivers and healthcare systems at large.5 There is, therefore, a strong public health incentive to choose safe therapies that provide increased health gains through improved vision and patient functioning, while offering an acceptable balance between benefits and costs. Ranibizumab (Lucentis®, Novartis Pharma AG, Switzerland) is a novel agent that is currently licensed for the treatment of visual impairment due to DME.6 Ranibizumab selectively inhibits active isoforms of human vascular endothelial growth factor A (VEGF-A) from binding to its receptors. VEGF-A stimulates growth of new blood vessels and is a major mediator of increased vascular leakage,7–10 mechanisms thought to be associated with retinal damage and progression of DME and the resulting visual impairment. The Phase III RESTORE trial enrolled 345 patients with visual impairment due to DME and assessed ranibizumab given either as monotherapy or in combination with laser photocoagulation, compared with laser photocoagulation alone. The results showed that ranibizumab alone or in combination with laser provided significantly greater improvements in best corrected visual acuity (BCVA) at 1 year compared with laser therapy alone, with mean average BCVA changes of +6.1 and +5.9 versus +0.8 letters, respectively.11 In addition, health-related quality of life, as assessed using the National Eye Institute Visual Function Questionnaire (NEI VFQ-25), was improved significantly from baseline for both ranibizumab treatment groups compared with laser therapy alone (p<0.05 for composite score and vision-related subscales). RESTORE is ongoing in a 2-year open-label extension phase. This report summarises the results of a health economic model that was developed based on RESTORE trial data to evaluate the cost-effectiveness of ranibizumab from a UK payer perspective when used as monotherapy or in combination with laser therapy compared with laser therapy alone. The model was developed separately from current assessments of ranibizumab by the UK Health Technology Assessment bodies, such as the National Institute for Health and Clinical Excellence. BODY.METHODS: A Markov model was constructed to simulate costs and changes in BCVA over a 15-year period in a hypothetical cohort of patients with DME (figure 1), based on data from the RESTORE trial (in which 87.5% of patients had type 2 diabetes and average baseline glycated haemoglobin was 7.3%). The average age at entry to the model was 63 years, consistent with patient characteristics in RESTORE. The model also assumed the same baseline BCVA distribution, but excluded patients with BCVA >75 letters, consistent with guidance from the ranibizumab summary of product characteristics that such patients may benefit less from treatment than those with baseline BCVA ≤75 letters.6 Figure 1Markov model structure. Health states are defined by best corrected visual acuity (BCVA) in the treated eye. Patients enter the model at treatment start where they are assumed to have BCVA as in RESTORE (>39 letters and ≤75 letters). BCVA is evaluated at 3-monthly intervals. After each cycle, patients may transition to any other health state including death; the probability of moving from health state A to health state B is based on RESTORE data (baseline to month 12) and literature. The model framework allocated eight linear health states defined by BCVA in the treated eye using a set of 10-letter (two-line) categories (table 1). Movement of patients from one health state to another was determined by transition probabilities that depended on the effectiveness of treatment and natural BCVA changes over time. A 10-letter range was used to categorise health states based on evidence that a 10-letter BCVA score loss is associated with a substantial decline in health-related quality of life (eg, loss of functional ability, increasing dependency, role limitations and impaired mental health).15 Each health state was assigned a quality-of-life index and cost. Costs and outcomes were accrued over 3-month cycles, applying half-cycle corrections. The time horizon in the base case was 15 years; although a lifetime horizon could be justified given the chronic nature of the condition, we selected a more conservative approach for the base case because of the lack of evidence on long-term prognosis. Table 1 Utility by BCVA in treated eye Health state defined by BCVA category (letters; treated eye) RESTORE * Lloyd et al 12 † Brown et al 13 § Mean utility (SE) 1: 86–100 0.860 (0.034) ‡ 0.830 0.839 2: 76–85 0.860 (0.014) 0.750 0.839 3: 66–75 0.813 (0.012) 0.750 0.783 4: 56–65 0.802 (0.014) 0.715 0.783 5: 46–55 0.770 (0.018) 0.680 0.732 6: 36–45 0.760 (0.027) 0.680 0.681 7: 26–35 0.681 (0.053) 0.530 0.630 8: 0–25 0.547 (0.083) 0.340 0.579 * Utility scores were calculated based on EQ-5D scores in RESTORE; EQ-5D scores were converted to utilities using social tariffs measured in a UK population. 14 Mean utility for each BCVA state was calculated using a regression technique for repeated measurements at baseline, month 3, month 6 and month 12. Data from several measurement points were pooled to cover all possible health state transitions with a sufficient sample size. A possible trend effect in the pooled data was rejected (p<0.05). † Patients underwent a Snellen visual acuity (VA) assessment and were categorised based on the better-seeing eye. Some adjustments were made to published values in order to convert VA ranges in Lloyd et al (obtained in a population of patients with diabetic retinopathy) to health states as defined in the current model. § Utilities were elicited from patients with diabetic retinopathy. Patients underwent a Snellen VA assessment and were categorised based on the better-seeing eye. Some adjustments were made to published values in order to convert VA ranges to health states as defined in the current model. ‡ Restricted to being greater than or equal to the utility in health state 2. BCVA, best corrected visual acuity. The key inputs and assumptions are summarised in table 2. Patients were assumed to receive ranibizumab treatment in year 1 at the frequency observed in RESTORE. In year 2, patients were assumed to need fewer injections, as observed in the Diabetic Retinopathy Clinical Research Network (DRCR.net) protocol I study (which included patients with comparable baseline demographics to RESTORE).1 A proportionately smaller number of monitoring visits was therefore assumed in year 2. After year 2, laser therapy was assumed to be administered as required in all arms, with no further need for ranibizumab; the assumed number of monitoring visits was further reduced accordingly. Table 2 Key model inputs and assumptions Time period Model input Combination therapy Ranibizumab monotherapy Laser monotherapy Source Year 1 BCVA progression RESTORE trial data, adjusted for drop-out rates RESTORE trial data, adjusted for drop-out rates RESTORE trial data, adjusted for drop-out rates RESTORE data on file Treatment frequency 7 injections + 2 laser sessions, as in RESTORE; drop-outs continue in standard care (ie, laser therapy) 7 injections; drop-outs continue in standard care (ie, laser therapy) 2 laser sessions; drop-outs continue in standard care (ie, laser therapy) RESTORE data on file Monitoring visits 12 12 4 SmPC and expert interview (data on file) Adverse events Negligible Negligible Negligible RESTORE data on file Year 2 BCVA progression Equal rates of improvement and worsening (3% in 3 months) Equal rates of improvement and worsening (3% in 3 months) Equal rates of improvement and worsening (3% in 3 months) Supported by DRCR.net and RESTORE data on file Treatment frequency 2 injections + 1 laser session 3 injections (no laser) 1 laser session Supported by DRCR.net Monitoring visits 8 10 4 Assumption Year 3 BCVA progression Constant rates of change of BCVA with a majority of patients having a decline in BCVA Constant rates of change of BCVA with a majority of patients having a decline in BCVA Constant rates of change of BCVA with a majority of patients having a decline in BCVA Calibrated to WESDR data (Supplementary Methods) Treatment frequency No additional ranibizumab; laser therapy as required No additional ranibizumab; laser therapy as required Laser therapy as required Assumption Monitoring visits 4 4 4 Assumption Any year Cost of blindness When BCVA ≤35 letters is reached in better-seeing eye When BCVA ≤35 letters is reached in better-seeing eye When BCVA ≤35 letters is reached in better-seeing eye Adapted from costing approach by Meads et al 16 (Supplementary table 2) Patient-level changes in BCVA data in RESTORE were used to derive transition probabilities in year 1. After year 1, long-term changes in BCVA were simulated assuming categorisation into one of three possible outcomes: ≥10 letters improvement within 3 months (one health state up), ≥10 letters worsening within 3 months (one health state down) or no change exceeding 10 letters within 3 months. BCVA, best corrected visual acuity; DRCR, Diabetic Retinopathy Clinical Research Network; SmPC, summary of product characteristics; WESDR, Wisconsin Epidemiologic Study of Diabetic Retinopathy. The average BCVA achieved in year 1 was assumed to be maintained during year 2, as was observed in the DRCR.net protocol I study.1 After year 2, all arms of the model followed natural disease history based on 4-year health state transition outcomes modelled from the Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR) reports.17–19 Transition probabilities were calibrated to adjust for the improvement in diabetes management since the WESDR reports (see Supplementary Methods), and predicted that around 30% of patients would be expected to exhibit a worsening in BCVA of at least 10 letters and 20% of patients would show an improvement of at least 10 letters over a 4-year time horizon (Supplementary table 1). Treatment discontinuation rates observed in RESTORE were applied to the model in year 1; it was assumed there would be no additional withdrawals from treatment in year 2. Adverse events were assumed to have a negligible impact on the cost-effectiveness of ranibizumab therapy based on the established safety profile of ranibizumab in clinical trials in DME and in wet age-related macular degeneration, an indication for which ranibizumab is also licensed.1 20–22 Mortality was estimated by adjusting general UK population death rates according to the increased RR of death in patients with DME. Mulnier et al estimated an increased mortality (HR 1.93) in a UK type 2 diabetes population relative to patients without diabetes,23 while Hirai et al estimated an HR of 1.27 for death in patients with clinically significant macular oedema (CSME) and diabetes relative to diabetic patients without CSME.24 We calculated a 2.45 RR of death in a DME population by multiplying these two ratios. Utility scores were calculated based on patient-reported outcomes data from RESTORE (table 2), in which patients completed the EuroQoL (EQ-5D) questionnaire at baseline and months 3, 6 and 12. Individual EQ-5D health scores were converted into utility scores using preferences from a UK population survey;14 mean utility scores were calculated for each health state (table 1). As these states were defined by BCVA in the treated eye, of which 67.2% were the worse-seeing eye at baseline, this method established an association between utility and BCVA changes in the treated eye. Health state costs included the costs of treatment and monitoring (Supplementary tables 3–6), and the costs associated with blindness (Supplementary table 2). Treatment costs included the costs of ranibizumab (Novartis UK, personal communication) and its administration, laser therapy and investigative procedures. Monitoring costs, including consultation and procedure costs, were estimated from the UK National Health Service Reference Costs.25 Costs of blindness included those incurred by the UK National Health Service for items such as low-vision aids, low-vision rehabilitation, residential or home care, depression and hip fracture/replacement as listed in the costing study by Meads and Hyde.16 Where older cost estimates were used, these were inflated to 2010 prices using the Hospital and Community Health Services index.26 The cost of blindness would be incurred only in patients reaching health states with BCVA ≤35 letters (Snellen ≤6/60) in the better-seeing eye. However, as the study assesses treatment response according to the enrolled eye, the proportion of patients reaching this level within the time horizon of the model is therefore uncertain. As such, the base case model adjusts for the cost of blindness on the basis of treated eyes reaching the BCVA ≤35-letter threshold. As with other model parameters subject to uncertainty, deviations from this assumption were explored in sensitivity analyses (Supplementary table 7). The main outcome measure was the incremental cost-effectiveness ratio (ICER), expressed as the additional cost per quality-adjusted life-year (QALY) gained by one treatment over another. An annual 3.5% discount rate was applied for future costs and utilities, consistent with the standard UK approach. Univariate sensitivity analyses assessed the uncertainty around specific data sources by exploring the effects on the ICER of inputs, as shown in table 3. Probabilistic sensitivity analysis assessed the overall uncertainty about the ICER based on variations in individual input parameters; details of the applied distributions and results are provided in Supplementary table 7. Table 3 Cost-effectiveness: base case and sensitivity analyses * Assumption/parameter Base case Sensitivity analyses Incremental cost Incremental QALY ICER ICER (% change) Ranibizumab monotherapy versus laser monotherapy  Base case – – £4191 0.17 £24 028 –  Discount rate of future costs and benefits 3.50% 0%–5% £3593–£4383 0.16 to 0.16 £17 051–£27 042 –29% to +13%  Time horizon 15 years 10–20 years £4738–£3991 0.14 to 0.19 £33 139 to £21 343 +38% to –11%  Cost of blindness £6477 –25% to +25% £4868–£3515 0.17 to 0.17 £27 907–£20 150 +16% to –16%  Long-term progression of VA Declining Constant or increasing £4487–£4693 0.17 to 0.17 £26 198–£28 413 +9% to +18%  Total number of ranibizumab injections 10 –4 injections to +4 injections £2171–£6774 0.17 to 0.17 £12 446–38 836 –48% to +62%  Baseline age 63 years 58 years £3767 0.20 £19 259 –20%  Source of utilities RESTORE Lloyd et al 12 £4191 0.22 £19 238 –20% RESTORE Brown et al 13 £4191 0.19 £21 953 –9% Combination therapy versus laser monotherapy  Base case – – £4695 0.13 £36 106 –  Discounting of future costs and benefits 3.50% 0%–5% £4271–£4828 0.16 to 0.12 £26 957–£40 096 –25% to +11%  Time horizon 15 years 10–20 years £5133–£4507 0.10 to 0.13 £49 294–£34 135 +37% to –5%  Cost of blindness £6477 –25% to +25% £5050–4340 0.13 to 0.13 £38 833–£33 378 +8% to –8%  Long-term progression of VA Declining Constant or increasing £5091–£5276 0.13 to 0.12 £40 852–£44 071 +13% to +22%  Total number of ranibizumab injections 9 –4 injections to +4 injections £3165–£7260 0.13 to 0.13 £24 340–£55 828 –33% to +55%  Baseline age 63 years 58 years £4393 0.15 £29 952 –17%  Source of utilities RESTORE Lloyd et al 12 £4695 0.16 £28 778 –20% RESTORE Brown et al 13 £4695 0.16 £29 576 –18% * Incremental cost measures the additional cost of ranibizumab monotherapy or combination therapy compared with laser monotherapy in the modelled time horizon (15 years in base case). Incremental QALY measures the corresponding QALY gain when ranibizumab monotherapy or combination therapy is compared with laser monotherapy. The ICER is calculated by dividing the incremental cost by the incremental QALY. The ICER is interpreted as the cost of achieving an additional year of life in perfect health. ICER, incremental cost-effectiveness ratio; QALY, quality-adjusted life-year; VA, visual acuity. BODY.RESULTS: The model predicted that after 1 year, a greater proportion of patients treated with ranibizumab monotherapy or combination therapy would have BCVA >65 letters (Snellen score >6/18) compared with patients treated with laser monotherapy (48% and 47%, respectively, versus 38% in the laser arm) (figure 2). After 15 years, the proportion with severe visual impairment in the treated eye (BCVA <35 letters, Snellen <6/60) was predicted to be 12% and 13% in the ranibizumab monotherapy and combination therapy groups, respectively, versus 19% in the laser group. Figure 2Modelled distribution by health states after (A) 1 and (B) 15 years. BCVA, best corrected visual acuity. Ranibizumab monotherapy was associated with an incremental gain of 0.17 QALY and cost of £4 191, corresponding to an ICER of £24 028 per QALY gained relative to laser monotherapy (table 3). Combination therapy provided an incremental gain of 0.13 QALY over laser monotherapy for an incremental cost of £4 695, leading to an ICER of £36 106 per QALY gained. Univariate sensitivity analyses showed that the model was stable and that ICERs were most sensitive to changes in the number of injections and time horizon (table 3). Using utilities elicited by Lloyd et al12 and Brown et al13 in patients with diabetic retinopathy led to greater QALY gains and lower ICERs for ranibizumab monotherapy or combination therapy relative to laser monotherapy. Probabilistic sensitivity analysis confirmed the model's robustness (Supplementary figure 1). Assuming a willingness-to-pay threshold of £30 000 per QALY gained, the probability that ranibizumab monotherapy would be cost-effective relative to laser monotherapy was estimated to be 64% (Supplementary figure 2); the corresponding probability for combination therapy relative to laser monotherapy was 42%. BODY.DISCUSSION: This is the first study to evaluate the cost-effectiveness of an anti-VEGF agent for the treatment of patients with DME causing visual impairment. Our economic model, which was based on data from the RESTORE clinical trial, shows that ranibizumab monotherapy provides superior improvements in visual acuity and is cost-effective relative to the current standard of care, laser photocoagulation. Ranibizumab monotherapy was associated with an ICER of £24 028 per QALY gained relative to laser therapy alone, a value within the willingness-to-pay threshold of £30 000 per QALY gained that has generally been considered cost-effective in the UK. The results of our model may be applicable to a broad spectrum of patients with visual impairment due to DME, as the RESTORE trial showed consistent efficacy of ranibizumab therapy across patient subgroups based on a range of demographic and disease characteristics. The cost-effectiveness of combined ranibizumab and laser therapy was less favourable than that of ranibizumab monotherapy (ICER £36 106 per QALY gained relative to laser therapy alone). While this in part reflects higher treatment costs, combination therapy also provided a lower predicted QALY gain than ranibizumab monotherapy (0.13 vs 0.17 QALY over the 15-year time horizon in the base case). A possible explanation for these results is the observation in RESTORE that combination therapy provided smaller improvements in BCVA in patients who had received prior laser therapy than those who had not (4.7 vs 6.9 letters gained, respectively).11 In RESTORE, the proportions of patients who received prior laser therapy were 52% (ranibizumab monotherapy), 47% (combination therapy) and 43% (laser therapy). Sensitivity analyses showed that the model results were robust to reasonable alterations in inputs and assumptions; ICERs were particularly sensitive to changes in the number of ranibizumab injections and the time horizon of the model. The base case assumed an average of 10 ranibizumab injections over 2 years, based on data from the DRCR.net protocol I study.1 Ranibizumab monotherapy remained cost-effective (ICER below £30 000 per QALY gained) up to a total of 13 injections. Increasing the number of injections beyond 13 resulted in an ICER outside the generally accepted threshold, emphasising that additional injections beyond 2 years of treatment will be an important cost driver. However, the current model is conservative in that it includes incremental costs for additional injections beyond year 2, but assumes no incremental benefit. The possible need for re-treatment beyond 2 years remains speculative; forthcoming data from the 2- and 3-year RESTORE follow-up and the DRCR.net 5-year data will improve our understanding of the likely duration of treatment. Longer time horizons would be expected to lead to improved cost-effectiveness of ranibizumab, because the benefits of improved vision accrue over time whereas treatment costs are incurred immediately once treatment is initiated. We have identified only one previously reported cost-effectiveness study of interventions for DME. Sharma et al modelled the cost-effectiveness of laser therapy alone for DME,27 comparing early and deferred laser treatment with no treatment based on 3-year outcomes from the Early Treatment Diabetic Retinopathy Study.28 The model included health states defined by BCVA and applied a 40-year time horizon based on a population with a mean age of 47 years. Over this time horizon, laser treatment was predicted to provide a gain of 0.236 QALY and was considered highly cost-effective for DME relative to no treatment. Our results cannot be compared directly with the Sharma et al27 study because RESTORE did not include a 'no treatment' arm; moreover, our model applied a 15-year time horizon from a baseline age of 63 years, consistent with RESTORE. However, if a baseline age of 47 years and time horizon of 40 years were applied to our model, as in the Sharma et al27 model, the RESTORE model would predict a 0.26 QALY gain and an ICER of £10 412 for ranibizumab monotherapy relative to laser therapy alone. This suggests that the cost-effectiveness of ranibizumab would be higher in patients with DME who were younger than the average in RESTORE (63 years) because of their longer life expectancy. Several methodological considerations should be noted. While treatment effectiveness was based on 1-year data from RESTORE, we projected long-term changes in BCVA using the best available clinical evidence. For year 2, the DRCR.net protocol I study supported the assumption that BCVA at 1 year was maintained on average to 2 years.1 BCVA changes in years 3–15 were based on reported progression in the WESDR study, which demonstrated a gradual decline in vision over time in DME. The rate of decline in BCVA was reduced by adjustment using data from both the Diabetes Control and Complications Trial and UK Prospective Diabetes Study to account for a less intensive systemic diabetes management regimen in the past relative to current practice.29–32 Sensitivity analysis showed that these assumptions had only a minor impact on the model results. Utilities were estimated from the BCVA of treated eyes in RESTORE, irrespective of whether this was the better-seeing or worse-seeing eye. This approach links utility values directly to the eye receiving the intervention. The RESTORE study protocol required the worse-seeing eye to undergo intervention unless there was a medical contraindication; 32.8% of treated eyes were the better-seeing eye at baseline, and 37.3% were the better-seeing eye at 12 months. Using the better eye as reference is supported by quality of life studies in vision-related conditions, which have shown that the better eye is the major driver of overall quality of life and patient functioning.33 Indeed, patient-preference studies have shown that the utility gains associated with treating the worse-seeing eye are uncertain, even though good vision in two eyes confers better quality of life than good vision in only one eye.34 As expected, subdividing the elicited utilities in RESTORE by better-seeing or worse-seeing eye showed that patients being treated in the better-seeing eye reported lower utility at a given level of BCVA than those treated in the worse-seeing eye. Nevertheless, utility measured in patients treated in the worse-seeing eye demonstrated significant sensitivity to variation in the BCVA in the treated eye, comparable with that of patients treated in the better-seeing eye. Unfortunately, the small sample of RESTORE patients in each BCVA health state meant that the resulting utility functions were not sufficiently robust to allow separate cost-effectiveness analysis by better-seeing or worse-seeing eye. It should also be stated that the option of not treating visual impairment in a better-seeing eye is not an ethical stance. We could not find published utility estimates from specific populations with DME. We performed sensitivity analysis using utilities reported in two studies based on populations with diabetic retinopathy (which includes a wider population than DME).12 13 Lloyd et al reported utilities elicited by the general population, while Brown et al reported utilities elicited by patients. This showed that applying the Lloyd et al12 utilities to the model greatly improved the cost-effectiveness of ranibizumab monotherapy, leading to a larger incremental gain of 0.22 QALY and a lower ICER of £19 238 per QALY gained relative to laser therapy alone. Applying the Brown et al13 utilities also increased the QALY gain with ranibizumab and led to an ICER of £21 953 per QALY gained for ranibizumab monotherapy versus laser therapy alone. The fact that the RESTORE utilities are less sensitive to BCVA decline may simply reflect the fact that a majority of the reference eyes in RESTORE were worse-seeing, while all reference eyes in the Lloyd et al12 and Brown et al13 studies were better-seeing. Limitations of this analysis should be considered. First, we modelled treatment of unilateral DME based on RESTORE data, but did not estimate the cost-effectiveness of treating bilateral DME. While 82.8% of patients in RESTORE had signs of bilateral DME at baseline, the proportion of patients who would have become eligible for treatment because of vision impairment in their fellow eye was not known. Treatment of both eyes may be relevant in many patients with bilateral DME, but there is a lack of evidence for the additional utility benefit of treating the fellow eye. Uncertainty also exists regarding the cost consequences of treating both eyes, where total cost is not likely to double given the possibility of achieving economies of scale from shared categorical spending, such as administrative and monitoring costs. An additional limitation was that the model assumed the cost of blindness would be incurred in patients reaching health states with BCVA ≤35 letters (Snellen ≤6/60) in the treated eye. There is also uncertainty inherent in working with published results of studies, as was necessary for incorporating the DRCR Network Protocol I results, as opposed to using patient-level data. While analysis using patient level data is clearly preferable, incorporation of findings from studies in addition to RESTORE, such as the highly comparable DRCR study, provide very useful data for answering critical questions relating to clinical practice in a broad spectrum of patients with DME. It should be noted, however, that some subgroups, such as patients with the poorest glycaemic control (high glycated haemoglobin), were excluded from these trials. In conclusion, the results of our economic model show that ranibizumab monotherapy is cost-effective relative to laser therapy alone in the treatment of DME causing visual impairment, while combined ranibizumab and laser therapy may be cost-effective depending on patient characteristics. The cost-effectiveness of ranibizumab monotherapy or combination treatment is expected to be higher in younger patients who have a longer life expectancy. These findings have important practical implications, given the high socio-economic burden of DME and the need for new, cost-effective treatments that reduce long-term progression to blindness. Ongoing studies, such as the RESTORE extension, will provide additional clarification of current uncertainties such as the need for ranibizumab injections after 2 years and the likelihood of recurrent DME. BODY.SUPPLEMENTARY MATERIAL: Supporting Statement Supporting Statement Supporting Statement Supporting Statement Supporting Statement Supporting Statement

TITLE: The efficacy of resiniferatoxin in prevention of catheter related bladder discomfort in patients after TURP - a pilot, randomized, open study ABSTRACT.BACKGROUND: Resiniferatoxin (RTX) has been shown to have variable efficacy in the treatment of intractable lower urinary tract symptoms and symptoms associated with neurogenic detrusor overactivity. It has also been used successfully in treating overactive bladder (OAB). In order to evaluate the potential and efficiency of RTX in treatment of catheter related bladder discomfort (CRBD) over post-operative period of transurethral resection of the prostate (TURP), we conducted the current pilot, randomized open study. ABSTRACT.METHODS: The study was comprised of 48 patients undergoing TURP in a single institute from September 2007 to September 2008. Patients were randomly divided into Group-RTX and Group-control. CRBD was classified into 4 degrees: degree 0, 1, 2, and 3. Patients with the most severed CRBD (degree-3) were treated with tolterodine. ABSTRACT.RESULTS: There were no statistical difference of patients' age, prostate volume, IPSS score, storage score and void score in IPSS, and QOL score between the two study groups(P=0.213, 0.356, 0.471, 0.554, 0.055 and 0.380, respectively). RTX pre-treatment reduced incidence of CRBD. 25% of the patients in RTX group had CRBD, at 6 hours/1 day and 3 days after TURP, significantly lower than the control group in which 75% of the patients during the same period (P=0.001). During the three days post-operative period, RTX also reduced severity of CRBD, compared with the control group. No patient in Group RTX sufferred CRBD of degree 3, a clear contrast to the control group in which 7 patients had degree 3 CRBD, during three days post-operative. Finally, less patients in RTX group required tolterodine, compared with control, P=0.009. Interestingly, the difference of CRBD incidence between Group RTX (50%) and control (75%) diminished 5 days after TURP, P=0.135. ABSTRACT.CONCLUSIONS: Pretreatment with intravesical resiniferatoxin significantly reduces the incidence and severity of catheter related bladder discomfort in patients after TURP. BODY.INTRODUCTION: Patients of benign prostatic hyperplasia undergoing transurethral resection of the prostate (TUR-P) often have urinary bladder catheterized. The catheter is frequently left in situ for the purpose of postoperative bladder drainage. As results of catheterization and lesions in proximal part of the urethra or bladder after TUR-P, patients often complain of bladder discomfort (an urge to void or discomfort in the suprapubic region) due to irritation from the catheterization over postoperative period. Severe symptoms of catheter related bladder discomfort (CRBD) are suprapubic pain and problems with outflow from the urethral catheter, conditions so-called bladder spasm. It is a result of lesions in the proximal part of the urethra or bladder due to the presence of indwelling urethral catheter (1). These symptoms are similar to symptoms of overactive bladder (OAB) (2). Muscarinic receptor antagonists oxybutynin and tolterodine which have been used successfully in the management of overactive bladder were also reported to be able to reduce catheter related bladder discomfort (3-7). According to the previous reports, these antagonists can reduce the incidence and severity of bladder discomfort in about half of the patients. However, the adverse events of muscarinic receptor antagonists (such as dry mouth, constipation, dysuria, and dyspepsia) have prevented them from being used as a routine management for CRBD (8-11). A number of investigators have tried other intervention methods, such as using intravesical diamorphine, oral opioids, or i.v. ketamine, to prevent CRBD (5,12). All of them reduce the incidence and severity of CRBD to some degree. However, the anaethetic nature of these drugs rendered them being less popular. As a potent capsaicin analogue, resiniferatoxin (RTX) has the capacity to excite and then to desensitize type C primary afferent fibers, which in turn leads to desensitization of bladder (13-16). RTX has been shown to have variable efficacy in the treatment of intractable lower urinary tract symptoms (LUTS) and symptoms associated with neurogenic detrusor overactivity (17,18). It has also been used successfully in treating OAB (14-19). However, RTX's efficacy on CRBD remains unknown. In order to evaluate the potential and efficiency of RTX in treatment of CRBD over post-operative period of TURP, we conducted the current pilot, randomized open study. BODY.METHODS.PATIENTS AND TREATMENT: Approval was received from the Institute's ethics committee and written informed consent was obtained from all the patients. The study was designed to be a pilot, randomized, open investigation. The study consisted of 48 consecutive patients who had undergone standard monopolar TURP at our institute from September 2007 to September 2008. Patients' prostate volume was measured by transrectal ultrasound (TRUS). Patients were also identified as bladder outlet obstruction according to results of urodynamics. Eligible patients were randomly divided. Twenty-four patients in RTX group (Group I) received, intravesically, 40 mL of 20 nM RTX (Hainan Newway Pharmaceutical Co., LTD, P.R. China) 20 hours before operation. Patients were asked to hold the drug for 30 minutes. Before introducing RTX, patients received intravesical instillation of 20 mL 2% lidocaine solution and 10 mL 0.75% bupivacaine solution, in order to prevent the temporary pain of bladder and urethra (20). Twenty-four patients in control Group (Group II) received no treatment before operation. After TUR-P, patients were indwelled with a three-way 22 Fr Foley catheter with 50 mL balloon, which was discharged 5 days after operation. All patients' age, prostates volume, IPSS (International Prostate Symptom Score), Storage Score in IPSS, Void Score in IPSS, and quality of life (QOL) score were collected. In Group I, the average age was 72+8.33 years (range from 56 to 88 years), the average prostates volume 64+19.06 mL (range from 32 to 92 mL), the average international prostate symptom score (IPSS) 22.7+3.16 (range from 16 to 28), the average Storage Score in IPSS 13.1+2.66 (range from 8 to 17), the average Void Score in IPSS 9.6+1.88 (range from 5 to 13), the average QOL score 4.2+0.88 (range from 2 to 6). In Group II, the average age was 69+6.93 years (range from 54 to 86 years), prostates volume 69+17.46 mL (range from 34 to 97 mL), IPSS 23.5+3.57 (range from 16 to 31), Storage Score in IPSS 12.6+2.66 (range from 8 to 17), Void Score in IPSS 10.7+2.21 (range from 6 to 14) QOL score 4.4+1.06 (range from 2 to 6). BODY.METHODS.ASSESSMENT OF CRBD: Catheter related bladder discomfort (CRBD) was assessed by the investigators at 6 hours, 1, 3 and 5 days after TURP. Severity of bladder discomfort was recorded as degree 0 (did not complain of any bladder discomfort), degree 1 (mild, reported by the patient only on questioning and does not need treatment), degree 2 (moderate, reported by the patient on their own, such as an urge to pass urine or discomfort in the suprapubic region, and does not need treatment), and degree 3 (severe, bladder spasm, reported by the patient without questioning, accompanied by behavioral responses) (4,5). All patients of degree 3 received tolterodine. BODY.METHODS.STATISTICS: The Independent samples t test and Fisher's exact test were used for statistical analyses of the different age, prostates volume, IPSS score and severity of CRBD between the Group I and Group II. SPSS 16.0 software was used to perform all statistical computations. BODY.RESULTS.GENERAL INFORMATION OF PATIENTS: There was no statistical difference between Group I and Group II in patients' age, prostates volume, IPSS score, Storage Score in IPSS, Void Score in IPSS, and QOL, P values were 0.213, 0.356, 0.471, 0.554, 0.055, and 0.380, respectively. BODY.RESULTS.EFFECT OF RTX ON CRBD: During the first three days of post-operative period, the incidence of patients' CRBD was lower in Group I than that in Group II (25% vs. 75%, P=0.001, Fisher's exact test). On the fifth day after surgery, although the incidence of CRBD was still lower in Group I than that in Group II (50% vs. 75%), there was no significant difference between them (P=0.135, Fisher's exact test). The incidence and severity of patients' CRBD was detailed in Tables 1 and 2. Table 1 Incidence of CRBD in Group I and II after TURP Group 6 h* 1 d* 3 d* 5 d I II I II I II I II Incidence of CRBD 25% (6/24) 75% (18/24) 25% (6/24) 75% (18/24) 25% (6/24) 75% (18/24) 50% (12/24) 75% (18/24) * The asterisk indicates P<0.01 between Group I and Group II. Table 2 Severity of CRBD in Group I and II after TURP Group 6 h 1 d 3 d 5 d I II I II I II I II Degree 0 18 6 18 6 18 6 12 6 Degree 1 4 6 4 8 4 11 10 12 Degree 2 2 5 2 10 2 7 2 6 Degree 3 0 7* 0 0 0 0 0 0 *Received tolterodine. No patient in Group-1 required muscarinic receptor antagonist (tolterodine). However, seven patients with degree 3 CRBD in Group-II received tolterodine during the first day after operation. Degree 3 symptoms in all these patients in Group II were controlled, and the degree of CRBD in these patients were reduced to lower grade after the treatment in days to follow. The number of patients who received tolterodine was statistically different between the two groups, P=0.009. Table 2 summarizes the details of severity of patients' CRBD in Group I and II. The intravesical instillation RTX caused temporal pain of bladder and urethra which were relieved without treatment. No other obvious adverse events were otherwise observed. BODY.DISCUSSION: Generally, the incidence of CRBD caused by catheter related bladder irritation during the postoperative period was about 55% (4,5). The incidence was much higher in patients after TUR-P (1). CRBD, especially bladder spasm not only results in severe suffering of the patients but also increase the chance of post-operative bleeding, clot forming, and may lead to a re-operation after TUR-P. Muscarinic receptor antagonists and anesthesia agents, such as oxybutynin, tolterodine, and ketamine have been used successfully for the management of CRBD (6,9-12). However, adverse event of these drugs, such as dry mouth, constipation, dyspepsia, dysuria, and urinary retention limited the use of these drugs post TUR-P (8-11). The post-TURP condition is therefore requires more efficient and safer management. In a reflex response, afferent input determines efferent output. Any approach which blocks the conduction of afferent nerve fibers may also be used to control CRBD. As a C fiber neurotoxin, resiniferatoxin (RTX) is an extract from the latex of Euphorbia resinifera. It is an ultrapotent capsaicin analog, and has a significant advantage of being less irritable. It binds to transient receptor potential vanilloid 1 (TRPV1), previously known as vanilloid receptor type 1 (VR1) (13,14,21). It has the capacity to excite and then to desensitize type C primary afferent fibers. Following the TRPV1 opening, the excitation leads to spike-like calcium currents. The transient high levels of intracellular calcium and other ions cause neuropeptide release (such as substance P) (22,23). The temporary bladder pain patients experienced during RTX treatment is known to be the result of detrusor contractions. In C afferent fibers, activation of bioelectricity will eventually stop and thus block bladder sensory transmission. The desensitization mechanisms by resiniferatoxin include: decreases in sensory input conveyed in bladder C-fibers, and decreases in the number of spinal neurons that become excited by bladder stimuli (24). There have been reported that RTX can be used to treat detrusor instability, BPH associated storage lower urinary tract symptoms, and overactive bladder (25). Thus, we hypothesized that intravesical administration of RTX may reduce the incidence and severity of CRBD after TUR-P. According the results of our study, the pre-operative administration of RTX intravesically could reduce both incidence and severity of CRBD during the first 3 days of post-operative period. The need for using antimuscarinic drugs was also reduced. In Group I, no patients had CRBD of degree 3. RTX was effective for detrusor instability, detrusor hyperreflexia, and alleviating bladder pain. In the US, most patients have indwelling catheter only 1-2 days after TURP. However, it may be companied severe lower urinary tract syndrome (LUTS). So, most patients usually indwelling catheter 3-5 days in China. Our result suggested that patients of RTX Group may not need catheter for 5 days either. However, patients will experience phasic detrusor contractions shortly after the intravesical instillation RTX. The detrusor contractions result in the patients having urge sensation to urinate. In addition, patients felt itching or warm in the lower abdomen during the initial period of RTX administration (26,27). All of these temporary discomforts can be prevented by intravesical administration lidocaine and bupivacaine solution. There was no patient suffering from severe adverse events in our study. In addition, in order to avoid intravesical instillation RTX related bladder discomfort overlapping with CRBD post-operation, we instilled RTX 20 hours before operation. Compared with instilling RTX half an hour before operation after patient received spinal anesthesia which we used in our preliminary study, the new procedure has the advantage of minimum waiting time prior and during anaesthesia, thus allowing less time to be spent in the operating theatre. On the other hand, the temporary discomfort related to RTX will have disappeared before the start of the operation. The 20-hour pre-operative period is therefore long enough for the temporary discomfort by RTX to diminish. The lowest dose to decrease bladder sensitivity and bladder pain was 10 nM. With an increase in dosage, significant improvement in symptoms and prolonged duration of improvement were obtained. In our study, with a dosage of RTX 20 nM, we achieved a significant reduction of incidence and severity of CRBD. The maximum effect was noted during the first three post-operative days. On the fifth day post-operative, the advantage of Group I over that of Group II no longer exists. Probably, with the recovery from lesions in proximal part of the urethra or bladder, the longer duration after TUR-P the more tolerable of patients become to indwelling urethral catheter. In additional, a portion of the patients in Group II had used tolterodine which would also reduce the incidence and severity of CRBD. Finally, we view that the dosage of RTX used in the present study was relatively low. A higher and safe dosage of RTX may reduce incidence and severity of CRBD more significantly and last longer time. On the other hand, it is proposed that intravesical instillation of RTX again on the third day after operation may be more effective for CRBD, and may be a useful adjunct for lower urinary tract symptoms post TUR-P. Clearly, more studies are necessary in testing different dosages and repeated intravesical instillation RTX. BODY.CONCLUSIONS: In our pilot randomized open study, a single intravesical administration of 40 mL of 20 nM RTX before TUR-P reduced both incidence and severity of post-operative CRBD via desensitization of bladder. The procedure has no contraindication and does not have other adverse event caused by RTX. The temporary pain of bladder and urethra can be prevented by analgesia with intravesical instillation of lidocaine and bupivacaine solution. Therefore, the procedure can be considered as a safe pretreatment option for decreasing CRBD in patients after TUR-P. Whether using a higher dose of RTX or repeated intravesical instillation RTX may reduce incidence and severity of CRBD more significantly require further investigation.

TITLE: Assessing the Effect of an Integrated Control Strategy for Schistosomiasis Japonica Emphasizing Bovines in a Marshland Area of Hubei Province, China: A Cluster Randomized Trial ABSTRACT.INTRODUCTION: More than 80% of schistosomiasis patients in China live in the lake and marshland regions. The purpose of our study is to assess the effect of a comprehensive strategy to control transmission of Schistosoma japonicum in marshland regions. ABSTRACT.METHODOLOGY/PRINCIPAL FINDINGS: In a cluster randomized controlled trial, we implemented an integrated control strategy in twelve villages from 2009 through 2011 in Gong'an County, Hubei Province. The routine interventions included praziquantel chemotherapy and controlling snails, and were implemented in all villages. New interventions, mainly consisting of building fences to limit the grazing area for bovines, building safe pastures for grazing, improving the residents' health conditions and facilities, were only implemented in six intervention villages. Results showed that the rate of S. japonicum infection in humans, bovines, snails, cow dung and mice in the intervention group decreased from 3.41% in 2008 to 0.81% in 2011, 3.3% to none, 11 of 6,219 to none, 3.9% to none and 31.7% to 1.7%, respectively (P<0.001 for all comparisons). In contrast, there were no statistically significant reductions of S. japonicum infection in humans, bovines and snails from 2008 to 2011 in the control group (P>0.05 for all comparisons). Moreover, a generalized linear model showed that there was a higher infection risk in humans in the control group than in the intervention group (OR = 1.250, P = 0.001) and an overall significant downward trend in infection risk during the study period. ABSTRACT.CONCLUSIONS/SIGNIFICANCE: The integrated control strategy, designed to reduce the role of bovines and humans as sources of S. japonicum infection, was highly effective in controlling the transmission of S. japonicum in marshland regions in China. ABSTRACT.TRIAL REGISTRATION: Chinese Clinical Trial Registry ChiCTR-PRC-12002405. BODY.INTRODUCTION: Schistosomiasis is an important public health issue in China where it continues to pose a serious threat to human well-being [1]–[3]. Since the People's Republic of China was established in 1949, the Chinese government has given high priority to the control of schistosomiasis, establishing a number of special bodies to manage control activities from the national to the township level [4]–[6]. These policies resulted in remarkable success in that the number of schistosomiasis patients were reduced by over 90%, from about 11.6 million cases in the 1950s to 726,000 cases in 2004 [7]. A further report in 2004 indicated that disease transmission had been interrupted or controlled in 42% of provinces, 40% of counties, and 53% of towns, previously endemic for schistosomiasis [8]. However, prospects for the control of schistosomiasis have been less optimistic in recent years, particularly since the termination of the World Bank Loan Project (WBLP) for schistosomiasis control at the end of 2001 [7]. Compared with the second national survey of S. japonicum in China in 1995, the third national survey conducted in 2004 showed that the prevalence of S. japonicum infection in humans had not substantially changed in the lake and marshlands and other areas of Southern China [7]. By the end of 2009, a total of 365,770 cases of S. japonicum were estimated in China, and 89 counties had not yet reached the criterion for transmission control which stipulated that the human prevalence should be less than 1% over a length of time [9], [10], i.e. the prevalence in these counties was >1%. Over the past 2–3 decades, the strategies for S. japonicum control in southern China, including the vast lake and marshland regions, has involved chemical mollusciciding, alteration of the oncomelanid intermediate snails habitats, and synchronous chemotherapy with praziquantel for all villagers and their bovines [6], [11]. Historically, these strategies achieved some effect, but recent studies demonstrated there were some problems as the control options resulted in environmental pollution leading to ecological damage [11], [12]. Moreover, owing to the high rates of reinfection in both humans and bovines, frequent flooding, and the complex environment, more persons have been infected and the habitat of the Oncomelania snails has increased in the lake and marshlands [11], [13]. Consequently, a more effective strategy was needed urgently in these areas. From 2005 through 2007, an important study of schistosomiasis japonica control was undertaken by Wang et al. in villages along the Poyang Lake in Jiangxi Province involving a comprehensive integrated approach aimed at reducing S. japonicum transmission to snails from cattle and humans, which play key roles as sources of S. japonicum [14]. The integrated strategy, which included removing bovines from snail-infested grasslands, providing farmers with mechanized farming equipment, building safe water systems, providing adequate sanitation, and implementing health education and synchronous chemotherapy with praziquantel for both villagers and bovines, was highly effective [14]. On the basis of identifying and controlling the main S. japonicum infection sources, we implemented a similar strategy (March 2009 through November 2011) to that employed by Wang et al. in the marshlands of Gong'an County [14], Hubei Province, another major endemic area for schistosomiasis japonica. The objective of the study was assessing the strategy's effect in marshland regions. BODY.METHODS: The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Protocol S1 and Checklist S1. BODY.METHODS.ETHICS STATEMENT: Written ethical approval for this study was obtained from the Ethics Review Committee of Hubei Provincial Center for Disease Control and Prevention (no. 200803). Written informed consent was obtained from all adults and from parents or guardians of minors before participation in the study. The participants had the opportunity to withdraw from the study at any time. Before beginning work on the study, the bovine owners provided consent to have their animals involved in the study. Moreover, permits for the bovines were obtained from Gong'an County Animal Husbandry Bureau. All animal work was carried out under the guidance of the Institute for Laboratory Animal Research (ILAR), and approved by Ethics Review Committee of Animal Experiments, Hubei Provincial Center for Disease Control and Prevention (no. 2008a05). Both doses of praziquantel (i.e., single 40 mg/kg dose for humans identified as stool egg-positive or 25 mg/kg for infected bovines) were within Chinese Ministry of Health published guidelines [15]. BODY.METHODS.STUDY AREA AND PARTICIPANTS: Gong'an County is located in typical marshland with a water area of 364 hectares (km2) along the mid-to-lower reaches of the Yangtze River in southwest Hubei Province, China. In 2009, there were 294 schistosomiasis-endemic villages (out of a total of 326), 539 cases of advanced schistosomiasis, and 36,612 cases of chronic schistosomiasis in Gong'an County; the prevalence of schistosomiasis in humans and bovines was 2.75% and 2.41%, respectively [16]. We carried out the study in 12 villages from 12 towns in Gong'an County which were selected by a two-stage random sampling procedure. The 12 towns were first randomly selected from 16 towns; then 12 schistosomiasis-endemic villages were selected from the 12 towns randomly (each town selected a village). Finally, these villages were randomly divided into intervention and control groups (Figure 1). E Jinghu, Gu Shengsi, Jianhong, Lianmeng, Tongqiao, Tuanjie were assigned to the intervention group and Guoqing, Nanyang, Qingyun, Qingji, Zhu Jiahu, Tongsheng were assigned to the control group (Figure 2). The villages had similar agriculture resources (rice, cotton and brassica napus) and the residents mainly were farmers (about 75%). The residents were exposed to contaminated water when they performed their agricultural or daily activities (i.e. cultivating crops, catching fish and washing clothes). 10.1371/journal.pntd.0002122.g001Figure 1Trial flowchart. 10.1371/journal.pntd.0002122.g002Figure 2Location of the study villages in the mid-to-lower reaches of the Yangtze River, Hubei province, China. The participants met the following inclusion criteria: a) must have been a resident of the village for more than 12 months; b) should be aged 6–65 years; c) should continuously reside in the village for the study period; d) have no serious diseases, such as malignant cancer. BODY.METHODS.THE COMPREHENSIVE STRATEGY TO CONTROL TRANSMISSION OF : Prior to implementing the new control strategy, routine interventions had been used in Gong'an County to control S. japonicum infection, which comprised synchronous praziquantel chemotherapy of both villagers and bovines and mollusciciding of snails [17], [18]. These procedures were continued in all 12 study villages for the duration of the study but additional interventions were incorporated into the intervention group. These comprised of the building of fences to limit the grazing area for bovines, building safe pastures for grazing, improving the residents' health conditions and facilities, and strengthening specialized schistosomiasis clinics at the village level. BODY.METHODS.THE COMPREHENSIVE STRATEGY TO CONTROL TRANSMISSION OF .FENCING AND SAFE PASTURES: Considering that bovines have been identified as the primary reservoir host of S. japonicum infection in China [6], [19]–[21], two measures were implemented (March to May, 2009) to reduce the influence of bovines on transmission. Firstly, 63 kilometres of fencing were installed to prevent animals from grazing in areas where Oncomelania snails were present. Warning signs were placed along the fenced areas, with the help of the local government authorities, and professional management staff were employed to ensure the intervention was effective. Secondly, 7 safe grazing pasture areas were established totaling 190 hectares (1,900,000 m2). Each area was 20–30 hectares and was designed to contain about 1,100 animals. The pastures were carefully selected with no or very few snails, which, if present, were quickly eliminated by mollusciciding. Specialized managers were employed to manage each of the grazing areas and ponds were established to enable the bovines to drink uncontaminated water. Fences comprised cement columns and barbed wire which ensured long-term usage. Each cement column was 160 centimeters (cm) in height and shaped as a triangular prism with 15 cm edges. Each column had three holes through which the barbed wire was fixed, and the distance between each was 2 meters. BODY.METHODS.THE COMPREHENSIVE STRATEGY TO CONTROL TRANSMISSION OF .IMPROVING HEALTH AND HEALTH CARE FACILITIES: To reduce the potential of humans as a source of infection for snails, measures were undertaken (May to July, 2009) to improve the health and well-being of villagers and the existing health care facilities. Safe water was provided to 2,826 households by building 8 new public wells or improving household wells already in place. To deal with humans feces, 1,545 home lavatories were constructed or repaired with three-cell septic tanks and 618 households were provided with marsh-gas pool latrines. Furthermore, all fishermen were provided with fecal-matter containers to prevent them from excreting feces directly into river or lake freshwater. BODY.METHODS.THE COMPREHENSIVE STRATEGY TO CONTROL TRANSMISSION OF .STRENGTHENING SPECIALIZED SCHISTOSOMIASIS CLINICS: In order to implement the interventions in the intervention villages, specialized schistosomiasis clinics were strengthened at the village level (March to May, 2009). As well as being responsible for managing the fences and the safety of the pastures, the clinic staff were responsible for examining the residents and livestock for schistosome infection, treating infected people or livestock with praziquantel, and providing health education on preventing S. japonicum infections and, in particular, in explaining the significance of bovines and humans as sources of S. japonicum infection for snails. BODY.METHODS.THE COMPREHENSIVE STRATEGY TO CONTROL TRANSMISSION OF .PRAZIQUANTEL CHEMOTHERAPY: During the study period, all participants were screened annually for S. japonicum infection by indirect hemagglutination assay (IHA), followed by fecal examination of IHA-positives using the Kato-Katz technique [15] (three thick smears). Kato-Katz-positives were treated with praziquantel at a single oral dose of 40 mg/kg. As well, all bovines were checked annually by the miracidium hatching test in dung [15]. All infected bovines were treated with a single dose of praziquantel at a dosage of 25 mg/kg body weight. BODY.METHODS.THE COMPREHENSIVE STRATEGY TO CONTROL TRANSMISSION OF .SNAIL CONTROL: Before implementing the new control strategy, mollusciciding using niclosamide had been conducted in all schistosomiasis-endemic villages (included all 12 study villages) in Gong'an County as part of the routine procedure. During the study period, a comprehensive approach, comprising mollusciciding with niclosamide and environmental modification, which mainly included using cement to change ditches' original environment, was used for snail control in all 12 study villages. Snail surveys were carried out twice-yearly (spring and autumn) along the river banks, ditches and marshlands around the villages [15]. An average of 110 hectares of snail habitat in the study villages were treated annually with molluscicide or environmental modification (89 hectares in 2008, 141 hectares in 2009, 134 hectares in 2010 and 74 hectares in 2011). BODY.METHODS.OUTCOME MEASURES: Five outcome measures, including the primary one (the prevalence of S. japonicum in humans) and four secondary ones (the rate of S. japonicum infection in bovines, cow dung, snails and mice), were determined annually and used to assess the strategy's effect. Participants that were positive for both IHA and Kato-Katz were defined as infected and the prevalence of S. japonicum of participants in all 12 study villages was in October/November, after the second transmission season. During the same period, the infection of S. japonicum of all bovines was checked in all 12 study villages by the miracidium hatching test in dung [15]. In addition, we gathered fresh cow dung from the grasslands which located in the 6 intervention villages (the sampled locations were the safe grazing pastures from 2009 to 2011), and used the miracidium hatching test to detect the infection of S. japonicum in cow dung. In April and May, we systematically sampled Oncomelania hupensis snails along the river banks and in marshlands and ditches around all 12 study villages. In every village, we investigated at least 2000 sample units (0.11 m2 frame) and gathered all the live snails in the frame, crushed them, and examined them for S. japonicum infection using microscopy. During the peak of the transmission season, July and August, we used exposure tests with mice to assess the infectivity of water in the 6 intervention villages [14]. Sentinel mice were exposed for 2 hours every day for 3 consecutive days. After 30 to 35 days, we sacrificed the mice and checked for adult worms of S. japonicum in their mesenteric veins. BODY.METHODS.STATISTICAL ANALYSIS: All statistical analyses were performed with the use of Statistics Analysis System, version 9.1 (SAS Institute Inc., Cary, NC, USA). Confidence intervals (CIs) were calculated using standard formulae based on the binomial distribution (annual infection rate of humans). Chi-square test or Fisher exact probability test were used to examine the differences of proportions. A generalized linear model (GLM) with a logit link and a binomial error distribution was used to analyze the risk of S. japonicum infection in humans. Generalized estimating equations of parameters with an unstructured variance-covariance matrix were used to account for repeated measures on individuals during the study period. The SAS proc GENMOD was used to estimate the parameters. Two-sided P-values were calculated for all tests and P-values <0.05 were considered statistically significant. BODY.RESULTS: Table 1 showed the characteristics of the participants in 2008. The differences of age between intervention and control groups were statistically significant (P<0.001). 10.1371/journal.pntd.0002122.t001 Table 1 Characteristics of the participants in intervention and control groups in 2008. Characteristics Control group (n = 5323) Intervention group (n = 5050) Age, years 38.4 (13.6) 39.4 (13.4) ** Sex ratio (F/M), n 2686/2637 2617/2433 Values are mean (SD) or number. ** P<0.001 for control group vs. intervention group. Figure 1 showed the flow diagram of the study design. In total, 10,373 participants were recruited for the study in 2008. The rates of loss to follow-up per year (2009–2011) for each village ranged from 1.1% to 18.5% in the intervention group and 2.1% to 25.2% in the control group (Table 2). The main reason for loss to follow-up was that the participants left the villages to seek work in urban areas. 10.1371/journal.pntd.0002122.t002 Table 2 Schistosoma japonicum infection in humans in intervention and control groups * . Village 2008 2009 2010 2011 N Infection No. Prevalence % (CI) N Infection No. Prevalence % (CI) N Infection No. Prevalence % (CI) N Infection No. Prevalence % (CI) Intervention group E Jinghu 535 19 3.55 (1.98, 5.12) 508 16 3.15 (1.63, 4.67) 452 10 2.21 (0.85, 3.57) 436 5 1.15 (0.14, 2.15) Gu Shengsi 1102 38 3.45 (2.37, 4.53) 1054 32 3.04 (2.00, 4.07) 997 19 1.91 (1.06, 2.76) 959 8 0.83 (0.26, 1.41) Jianhong 765 23 3.01 (1.79, 4.22) 718 17 2.37 (1.25, 3.48) 685 12 1.75 (0.77, 2.74) 637 4 0.63 (0.01, 1.24) Lianmeng 994 38 3.82 (2.63, 5.02) 964 31 3.22 (2.10, 4.33) 892 19 2.13 (1.18, 3.08) 885 8 0.90 (0.28, 1.53) Tongqiao 1021 33 3.23 (2.15, 4.32) 1001 24 2.40 (1.45, 3.35) 990 21 2.12 (1.22, 3.02) 963 8 0.83 (0.26, 1.41) Tuanjie 633 21 3.32 (1.92, 4.72) 626 18 2.88 (1.56, 4.19) 611 12 1.96 (0.86, 3.07) 587 3 0.51 (−0.07, 1.09) Total § 5050 172 3.41 (2.91 ,3.91) 4871 138 2.83 (2.37 ,3.30) 4627 93 2.01 (1.61 ,2.41) 4467 36 0.81 (0.54 ,1.07) Control group Guoqing 788 26 3.30 (2.05, 4.55) 755 21 2.78 (1.61, 3.96) 737 22 2.99 (1.75, 4.22) 716 16 2.23 (1.15, 3.32) Nanyang 1035 37 3.57 (2.44, 4.71) 967 30 3.10 (2.01, 4.20) 921 26 2.82 (1.75, 3.89) 914 23 2.52 (1.50, 3.53) Qingyun 894 26 2.91 (1.80, 4.01) 812 23 2.83 (1.69, 3.98) 750 21 2.80 (1.62, 3.98) 748 20 2.67 (1.52, 3.83) Qingji 963 32 3.32 (2.19, 4.46) 889 28 3.15 (2.00, 4.30) 822 25 3.04 (1.86, 4.22) 818 23 2.81 (1.68, 3.95) Zhu Jiahu 712 22 3.09 (1.82, 4.36) 697 17 2.44 (1.29, 3.59) 683 16 2.34 (1.21, 3.48) 595 16 2.69 (1.39, 3.99) Tongsheng 931 27 2.90 (1.82, 3.98) 807 22 2.73 (1.60, 3.85) 727 18 2.48 (1.34, 3.61) 696 16 2.30 (1.18, 3.41) Total # 5323 170 3.19 (2.72, 3.67) 4927 141 2.86 (2.40 ,3.33) 4640 128 2.76 (2.29 ,3.23) 4487 114 2.54 (2.08 ,3.00) * CI denote 95% confidence intervals. § As compared with 2008 in the intervention group, there was a statistically significant change in the rate of infection in 2010 and 2011 (P<0.001), and there was no statistically significant change in 2009 (P = 0.101). # As compared with 2008 in the control group, there was no statistically significant change in the rate of infection from 2009 to 2011 (P = 0.328, P = 0.204, P = 0.055, respectively). BODY.RESULTS.INFECTION IN HUMANS: The prevalence of S. japonicum in humans was 3.41%, 2.83%, 2.01%, and 0.81% from 2008 to 2011 in the intervention villages (comparing with 2008, P = 0.101, P<0.001 and P<0.001, respectively, Table 2). In the control villages, there was no significant statistically decline in the rate of infection from 2008 to 2011 (P>0.05 for all comparisons). The generalized linear model (Table 3), yielding odds ratios (OR) adjusted for participants' age and gender, showed a higher infection risk in humans in the control villages than the intervention villages (OR = 1.250, P = 0.001); and an overall significant downward trend in infection risk during the study period. 10.1371/journal.pntd.0002122.t003 Table 3 Analyzing the risk of Schistosoma japonicum infection in humans by generalized linear model * . Variables OR (95% CI) P-value Intercept 0.012 (0.009, 0.016) <0.001 Age 1.004 (0.999, 1.009) 0.091 Gender Female 1.047 (0.919, 1.193) 0.492 Male 1.000 - Study year 2008 2.026 (1.670, 2.460) <0.001 2009 1.734 (1.417, 2.123) <0.001 2010 1.436 (1.166, 1.768) <0.001 2011 1.000 - Group Control group 1.250 (1.095, 1.427) 0.001 Intervention group 1.000 - * The model includes participants' age, gender, study year, group. BODY.RESULTS.INFECTION IN BOVINES: The S. japonicum infection rates in bovines were 3.3%, 2.9%, 1.3%, and 0.0% from 2008 to 2011 in the intervention villages (comparing with 2008, P = 0.820, P = 0.111 and P<0.001, respectively, Table 4). In contrast, there was no significant decline in the rate of infection during the 4-year period in the control villages (P>0.05 for all comparisons). 10.1371/journal.pntd.0002122.t004 Table 4 Schistosoma japonicum infection in bovines in intervention and control groups * . Year Intervention group Control group N Infection No. (%) N Infection No. (%) P-value a P-value b P-value c 2008 305 10 (3.3) 345 11 (3.2) - - 1.000 2009 309 9 (2.9) 337 9 (2.7) 0.820 0.821 1.000 2010 311 4 (1.3) 325 8 (2.5) 0.111 0.646 0.385 2011 318 0 (0.0) 313 8 (2.6) <0.001 0.650 0.004 * Statistical method was the Fisher exactly probability test. a Compared with 2008 in the intervention group. b Compared with 2008 in the control group. c Compared between the intervention and control groups from 2008 to 2011. BODY.RESULTS.INFECTION IN SNAILS: From 2008 to 2011, 11 of 6,219, 9 of 5,975, none of 25,010, and none of 15,490 snails were infected with S. japonicum in the intervention villages (comparing with 2008, P = 0.824, P<0.001 and P<0.001, respectively, Table 5). In the control villages, there was no significant statistically decline in the rate of infection from 2008 to 2011 (P>0.05 for all comparisons). 10.1371/journal.pntd.0002122.t005 Table 5 Schistosoma japonicum infection in snails in intervention and control groups * . Year Intervention group Control group N Infection No. N Infection No. P-value a P-value b P-value c 2008 6,219 11 4,574 9 - - 0.824 2009 5,975 9 8,664 6 0.824 0.054 0.187 2010 25,010 0 11,889 13 <0.001 0.231 <0.001 2011 15,490 0 9,493 8 <0.001 0.116 <0.001 * Statistical method was the Fisher exactly probability test. a Compared with 2008 in the intervention group. b Compared with 2008 in the control group. c Compared between the intervention and control groups from 2008 to 2011. BODY.RESULTS.INFECTION IN COW DUNG: In the intervention villages in 2008, 21 of 533 (3.9%) of the cow dung samples contained S. japonicum eggs. After the implementation of the new control strategy, 15 of 483 (3.1%), 5 of 476 (1.1%) and none of 356 cow dung were infected in 2009, 2010 and 2011, respectively (comparing with 2008, P = 0.501, P = 0.005 and P<0.001, respectively). BODY.RESULTS.INFECTIVITY OF WATER: In 2008, before implementation of the new control strategy, 19 of 60 (31.7%) mice were infected with S. japonicum. In 2009, 2010 and 2011, 13 of 60 mice (21.7%), 7 of 120 (5.8%) and 2 of 120 (1.7%) mice were infected, respectively (comparing with 2008, P = 0.216, P<0.001 and P<0.001, respectively). BODY.RESULTS.ADVERSE EVENTS: No serious adverse events were reported in the study. BODY.DISCUSSION: Over the past few years, investigations showed that more than 80% of S. japonicum patients in China were found in the lake and marshland areas of Hunan, Hubei, Jiangxi, Anhui, and Jiangsu provinces [22]. Therefore, schistosomiasis control in these regions was especially important. Because of high rates of S. japonicum reinfection in both humans and bovines in marshland and lake regions [21], [23], [24], it was very difficult to reduce the rate of S. japonicum infection in humans to a relatively low level (such as less than 1%). The study of Wang et al. showed that a comprehensive control strategy, which was based on interventions to reduce the transmission of S. japonicum infection from cattle and humans to snails, can solve this problem [14]. However, the authors mentioned that their study included only a small number of villages and the villages were not selected in a random manner. So they were not sure whether the strategy was still highly effective in other endemic areas. A similar study was implemented in Jiangsu Province of China, but this study did not have parallel control groups [25]. In our study, we applied a similar strategy but strived to avoid these limitations. We adopted a random manner to select 12 villages in Gong'an County, Hubei Province and randomly assigned them to intervention or control groups. Our study proved that the new comprehensive strategy to control transmission of S. japonicum was highly effective in the study areas. Firstly, the prevalence of schistosomiasis in humans declined to a relatively low level after implementing the new interventions. Compared with 2008, the rate of infection in humans in the intervention villages decreased from 3.41% in 2008 to 0.81% in 2011. The generalized linear model showed that a higher infection risk for humans in the control villages than in the intervention villages (OR = 1.250, P = 0.001). Secondly, comparing with 2008, the rate of infection in bovines, snails and cow dung in the intervention group decreased from 3.3% to none, 11 of 6,219 to none, and 3.9% to none in 2011, respectively. Thirdly, during the peak transmission period, after the implementation of the new control strategy, the rate of infection in sentinel mice decreased from 31.7% in 2008 to 1.7% in 2011. It should be noted that the randomized, controlled design pertains only to the human, bovine, and snail data while the environmental cow dung and infectious water data were only collected in the intervention villages. Bovines are the main infection source of S. japonicum in the marshlands and lakes in China, and are responsible for an estimated 75%–90% of the egg contamination [20], [21]. Therefore, it was very important to reduce the role of bovines as an infection source. Recently, the main measures included providing farmers machines instead of bovines and prohibiting bovines in these regions. These measures were effective [14]. However, if these measures were used in some rural areas of Hubei Province, there would be some challenges. Firstly, the machines are not suitable due to the limitation of landform or farmers' knowledge. Secondly, the bovines can bring additional economic benefits for villagers, so the villagers were reluctant to give up cattle rearing. For these reasons, in our study we adopted new interventions for bovines which included building safe pastures for grazing and preventing bovines from grazing on snail habitat beach areas. Our study proved that these measures were effective. Moreover, these measures were sustainable because the cost of fences and pastures was a one-time expense because the material is durable. We used a triangular cement fence post, which requires less cement than a square shape. Our study showed that there was no statistically significant decline in the S. japonicum infection prevalence in humans, bovines and snails from 2008 to 2011 in the control villages. Past studies showed that the effect of chemotherapy with praziquantel for humans and cattle was limited in controlling schistosomiasis in lake and marshland regions [17]–[19], because it does not prevent reinfection [23]–[25]. In contrast, the integrated control strategy can reduce the transmission of S. japonicum infection from humans and cattle to snails, reducing contaminated water as a source of infection in humans [14]. However, this difference is of little practical consequence unless the integrated interventions lead to more sustainable control and a reduction of the basic reproductive number (R0). If R0 can be kept less than 1 over a length of time, the disease could be gradually eliminated [26]. This is probably the most important potential outcome of the intervention. However, a longer term follow-up study is needed to test this potential outcome. Taken together, we considered that the new interventions, especially the interventions for bovines and humans, were the most important components of the integrated control strategy. A potential limitation of the study was that we did not investigate the status of S. japonicum infection in people who quit the study. Thus, we could not compare the difference of these people's infection between intervention and control groups. BODY.DISCUSSION.CONCLUSIONS: An integrated control strategy, aiming to weaken the roles of bovines and humans as sources of S. japonicum infection, was effective in controlling the transmission of S. japonicum in marshland regions of China. BODY.SUPPORTING INFORMATION: Checklist S1CONSORT Checklist.(DOC)Click here for additional data file. Protocol S1Trial protocol (Chinese language file).(DOC)Click here for additional data file.

TITLE: Comparison of the Effects of Maternal Supportive Care and Acupressure (at BL32 Acupoint) on Labor Length and Infant's Apgar Score ABSTRACT.BACKGROUND AND OBJECTIVES:: Prolonged labor leads to increase of cesarean deliveries, reduction of fetal heart rate, and maternal as well as infantile complications. Therefore, many women tend to use pharmacological or non-pharmacological methods for reduction of labor length. The present study aimed to compare the effects of maternal supportive care and acupressure (at BL32 acupoint) on labor length and infant's Apgar score. ABSTRACT.METHODS:: In this clinical trial, 150 women with low-risk pregnancy were randomly divided into supportive care, acupressure, and control groups each containing 50 subjects. The data were collected using a questionnaire including demographic and pregnancy characteristics. Then, the data were analyzed using Chi-square test and one-way ANOVA. ABSTRACT.RESULTS:: The mean length of the first and second stages of labor was respectively 157.0±29.5 and 58.9±25.8 minutes in the supportive care group, 161.7±37.3 and 56.1±31.4 minutes in the acupressure group, ad 281.0±79.8 and 128.4±44.9 minutes in the control group. The difference between the length of labor stages was significant in the three study groups (P<0.001). Moreover, the frequency of Apgar score≥8 in the first and 5th minutes was higher in the supportive care and acupressure groups compared to the control group, and the difference was statistically significant (P<0.001). ABSTRACT.CONCLUSION:: Continuous support and acupressure could reduce the length of labor stages and increase the infants' Apgar scores. Therefore, these methods, as effective non-pharmacological strategies, can be introduced to the medical staff to improve the delivery outcomes. BODY.1. INTRODUCTION: Delivery pain is an acute pain which rapidly increases and is affected by physiological, psychological, social, cultural, and environmental factors (Leeman et al., 2003). Excessive pain intensifies mother's fear and anxiety during delivery and stimulates sympathetic nervous system. These, in turn, enhance secretion of catecholamines, such as epinephrine and norepinephrine, eventually leading to more pain, prolonged labor stages, and dissatisfaction with the delivery experience (Sercekus & Okumus, 2007). Prolonged labor results in anxiety, fear, and fatigue which play a major role in reduction of mother's self-confidence and self-esteem. Thus, the women experiencing prolonged labor tend to make use of analgesic methods. Prolonged labor also increases the probability of damage, prenatal mortality, utilization of oxytocin, and rate of cesarean and instrumental delivery (Kaptchuk, 2002; May& Elton, 1998; Rabl, Ahner, Bitschnau, Zeisler, & Husslein, 2001). Furthermore, the relationship between chronic stress and delivery outcomes indicates the necessity of interventions for reducing this factor (Fink et al., 2011). Nowadays, various pharmacological and non-pharmacological methods are used for decreasing labor pain. Yet, since pharmacological methods might be accompanied by some complications for both mother and fetus, non-pharmacological ones are more welcomed. Up to now, a large number of non-pharmacological methods have been proposed for reduction of labor pain with acupressure and supportive care being two important ones (Kimber et al., 2008). Acupressure is a comprehensive treatment method which dates back to 5000 years ago. In this method, similar to acupuncture, specific reflex points on the body are used for treatment. By pressing these points, muscle tension is removed and blood circulation and vital energy are improved (Yang, 2001). Some researchers believe that reduction of pain following stimulation of acupoints is due to the fact that it prevents transfer of pain stimulants and increases the blood endorphin levels (Chung, Hung, Kuo, & Huang, 2003). Park et al. (2003) stated that acupressure increased the intensity of uterine contractions (Park, Cho, Kwon, Ahn, Lim, & Chang, 2003). In the same line, Skilnand et al. (2002) showed that the first stage of labor was shorter among the participants who underwent acupressure (Skilnand, Fossen, & Heiberg, 2002). Overall, various acupoints are employed for induction and control of delivery and BL32 (Ciliao) is one of these points (Cook & Wilcox, 1997). The other non-pharmacological method used in the current study was supportive care. This method involved continuous presence of doula and provision of psychological support (reassuring, encouraging, and guiding the mother), physical support (palpation, massage, coldness, hotness, hydrotherapy, position change, and movement), informing and guiding the mother, and facilitation of creation of relationship (helping the woman to express her needs) (Simkin & Bolding, 2004). According to most doulas, mothers cannot predict how labor affects them because they do not know about the delivery process and judge themselves negatively (Gilliland, 2011). On the other hand, self-confidence and the ability to adapt with labor are the predictors of labor pain experience. By supportive care, women can successfully cope with labor pain and stress and feel strong and mentally calm (Simkin & Bolding, 2004). In the study by Kennel et al. (1991), doula's continuous support reduced the labor length by 1-2 hours and increased the mother's capability to control laborthereby resulting in a positive delivery experience (Kennel, Klaus, McGrath, Robertson, & Hinkley, 1991). Considering the effects of prolonged labor on the delivery outcomes, the present study aims to compare the effects of maternal supportive care and acupressure (at BL32 acupoint) on labor length and infant's Apgar score. BODY.2. METHODS.2.1 STUDY DESIGN: This randomized clinical trial was conducted in the delivery ward of the selected educational center of Shiraz University of Medical Sciences (Shoushtari hospital in Iran) in 2012. BODY.2. METHODS.2.2 SETTING AND SAMPLE: Considering d=5, α=0.05, 1-β=0.90, SD=7, and the following formula, a 126-subject sample size was determined for the study (42 in each group). However, due to the possibility of loss, the sample size was increased to 150 subjects (50 in each group): Then, the subjects were selected through simple random sampling and were divided into supportive care, acupressure, and control groups using stratified block randomization. In doing so, a number was randomly selected from the table of random numbers and the researcher moved toward the right or left column or row and wrote the 5 digit numbers down. Since the participants were divided into 3 groups in this study, 3-therapy method was used and classification was performed as follows: A: supportive care group, B: acupressure group, and C: control group. Accordingly, ABC: 1, ACB: 2, BAC: 3, BCA: 4, CAB: 5, and CBA: 6. It should be noted that numbers 0, 7, and 9 were ignored. BODY.2. METHODS.2.3 ETHICAL CONSIDERATIONS: Ethical Committee Approval Code in Medical Research, Shiraz University of Medical Sciences in Iran is CT-P-4985. BODY.2. METHODS.2.4 MEASUREMENTS: The inclusion criteria of the study were being primiparous or multiparous, being physically and mentally healthy, having at least diploma, being 18-35 years old, singleton pregnancy, cephalic presentation, gestational age of 37-42 weeks, 4cm dilation, and having at least 2-3 uterine contractions in 10 minutes. After signing written informed consents, the selected women were divided into acupressure, supportive care, and control groups through permuted block randomization. The women with preeclampsia, induced labor, non-cephalic presentation, cephalopelvic disproportion, multiple birth, and those who smoked, suffered from underlying diseases, and were unwilling to take part in the study were excluded from the research. BODY.2. METHODS.2.5 PROCEDURE: In the supportive care group, the researcher as the doula accompanied the mother since hospitalization up to delivery. Emotional supports during labor included palpation, kindly massaging the mother, and reassuring her. The doula also provided the mother with information about the origin of pain and process of delivery. Besides, physical support included helping the mother to change her position and move during labor. In the acupressure group, in 3-4 and 7-8cm dilation, the participants were located in the appropriate position and BL32 acupoint was pressed. This acupoint is located in the second hole of sacral bone (16). The pressure was continuously and gently applied by both thumbs for 30 minutes (Figure 1). Before sampling, the researcher was trained regarding performance of acupressure by a physical medicine and rehabilitation specialist, so that equal pressure was applied in each performance. After the training, the pressure applied by the right and the left thumb was measured as 1405 and 1277 mmHg, respectively. The pressure was applied by the beginning and stopped at the end of the contractions. Since the interventions were not performed continuously and the researcher took a rest during the contractions, no problems were faced for application of pressure. Figure 1Location of the BL 32 point; Reference: http://acupunctureschoolonline.com/bl-31%E2%80%93bl-34-eight-liao-baliao-acupuncture-points.html The control group only received the hospital's routine care services. After the interventions, the three study groups were compared regarding the length of the first and second stages of labor as well as the infants' Apgar scores. BODY.2. METHODS.2.6 DATA ANALYSIS: The data were analyzed using Chi-square test and one-way ANOVA. Post-hoc test was also used to identify the groups with significant differences. BODY.3. RESULTS: The results of Chi-square test showed no significant difference among the three groups regarding age distribution and the participants' mean age (P=0.496). Also, no significant difference was found among the three groups concerning the mothers' level of education (P=0.584) and occupation (P=0.781). Additionally, the participants' mean gestational age was 38.9±1.1 weeks and the results of one-way ANOVA indicated no significant difference among the three groups regarding the mean gestational age (P=0.158). According to the results of one-way ANOVA, the mean length of the first stage of labor was higher in the control group (281.0±79.8) compared to the supportive care (157.0±29.5) and acupressure groups (161.7±37.3). Thus, the control group's mean length of the first stage of labor was 124.0 minutes higher compared to the supportive care group and 119.3 minutes higher compared to the acupressure group, and the difference was statistically significant (P<0.001). Also, the mean length of the second stage of labor in the control group was 69.5 minutes higher compared to the supportive care group and 72.3 minutes higher compared to the acupressure group, and the difference was statistically significant (P<0.001). According to the results of post-hoc and Least Significant Difference (LSD) tests in the first and second stages of labor, a significant difference was found between the supportive care and the control group as well as between the acupressure and the control group. Overall, the mean length of the first and second stages of labor was lower in the two intervention groups in comparison to the control group. The lowest mean lengths of the first and second stages of labor (157.0±29.5 and 58.9±25.8, respectively) were related to the supportive care group, while the highest mean lengths (281.0±79.8 and 128.4±44.9, respectively) were observed in the control group (Table 1). Table 1 Comparison of the mean duration of the first and second stages of labor in the intervention and control groups stage of labor Group stage of labor Group (n=50) P value Supportive care Acupressure Control First stage Duration (min) M±SD 157.0±29.5 161.7±37.3 281.0±79.8 0.000 Maximum 210 240 560 First stage Minimum 100 60 170 CT Upper limit 165.3 172.3 304.2 Lower limit 148.6 151.0 258.8 Duration (min) M±SD 58.9±25.8 56.1±31.4 128.4±44.9 Second stage Maximum 130 180 200 0.000 Minimum 20 15 50 CT Upper limit 66.2 65.0 141.1 Lower limit 51.5 47.1 115.6 Significance level: P<0.05. SD: standard deviation. The results of Chi-square test revealed a significant difference among the three groups regarding the first- and fifth-minute Apgar scores (P<0.001). As Table 2 depicts, the frequency of first-minute Apgar scores<8 in the control group was higher compared to the supportive care and acupressure groups by 46% and 34%, respectively. Also, the frequency of fifth-minute Apgar scores<8 in the control group was higher compared to the supportive care and acupressure groups by 20% and 18%, respectively. The frequency of first-minute Apgar scores≥8 was higher in the acupressure and supportive care groups (86% and 74%) compared to the control group (40%). The frequency of fifth-minute Apgar scores≥8 was also higher in the acupressure and supportive care groups in comparison to the control group (98%, 96%, and 78%, respectively) (Table 2). Table 2 Comparison of the first and fifth minute Apgar scores of the infants in the intervention and control groups Stage of labor Group (n=50) P value Supportive care Acupressure Control Apgar score after birth <8 7(14) 13(26) 30(60) 0.000 First minute apgar score ≥8 43(86) 37(74) 20(40) Apgar score after birth <8 1(2) 2(4) 11(22) 0.000 Five minute apgar score ≥8 49(98) 48(96) 39(78) Significance level: P<0.05. SD: standard deviation. BODY.4. DISCUSSION: The findings of the present study revealed a significant difference between the supportive care as well as the acupressure group and the control group regarding the mean length of the first and second stages of labor (P<0.001). In fact, the mean length of the first and second stages of labor in the control group was respectively 124 and 69.5 minutes higher compared to the supportive care group. Providing the mother with psychological and emotional support is one of the dimensions of supporting the mother by the doula. In this respect, the findings of the current study were in line with those of the study by Kennel et al. (1991). In that study, women who had experienced delivery acted as doulas and the results indicated a 25% decrease in the labor length in the supported group compared to the control group. A large number of studies have also shown a reduction in the labor length in the supported women compared to those receiving hospital's routine care. This reduction was reported as 2.8 hours in the study by Zhang et al. (1996), 44 minutes in the study by Scott (Scott, Berkowitz, & Klaus, 1999), and 0.58 hours in the study by Hodnett (Hodnett, Gates, Hofmeyr, Sakala, & Weston, 2011). In the study conducted by Campell, the mean length of the first stage of labor was 10.4±4.3 hours in the supported group and 11.7±4.8 hours in the control group. In addition, the mean length of the second stage of labor was 58±51 and 64±57 minutes in the supported and the control group, respectively (Campell, Lake, & Falk, 2006). Similarly, Longer showed that the mean length of labor was 4.56 hours in the supported group and 5.58 hours in the control group (Langer, Campero, Garcia, & Reynoso, 1998). Also, Hofmeyr et al. (1991), Chalmers (1993), Klaus and Kennell (1997), and Pascali-Bonaro (Pascali-Bonaro & Kroeger, 2004) reported that the mean length of labor was lower in the supported group in comparison to the control group. The findings of all the aforementioned studies were in agreement with those of the present study. In all these studies, doulas accompanied the mothers since hospitalization up to the delivery and supported her psychologically. According to these researchers, supporting the mother during delivery can lead to considerable changes in the delivery process, including modification of uterine function, improvement of uterine contractions, creation of effective contractions, and reduction of labor length. On the contrary to the results of the current study, Bruggemann showed that the mean length of the first stage of labor was 3.4 hours in the supported group and 3.8 hours in the control group (Bruggemann, Parpinelli, Osis, Cecatti, & Neto, 2007). Similarly, Bruggemann and McGrath (McGrath & Kennell, 2008) revealed no significant difference between the intervention (presence of doula) and the control group regarding the mean length of labor. The difference between the results of these studies and the present one might be due to the fact that they were conducted on the individuals from high social levels and the study participants could take their family members to the delivery room either with or without the doula. Therefore, both groups were highly supported and the effect of presence of doula could not be truly investigated. Overall, presence of doula before the delivery, encouragement, consolation, and palpation of the women, and suggestion of positions which are effective in fetal descent, increase production of oxytocin, enhance women's threshold of pain, and modify delivery pain patterns, thereby decreasing the labor length (Taylor, Klein, Lewis, & Gruenewald, 2000). According to the western medicine perspective, acupressure can create balance during delivery, reduce delivery pain, and improve the delivery process by increasing the uterine contractions (Beal, 1992). Acupressure is a simple, inexpensive non-pharmacological method for controlling delivery. In general, various acupoints are used for induction and control of delivery (Cook & Wilcox, 1997). In the present study, acupressure was performed at BL32 acupoint and the results revealed a 119.3 minute reduction in the first stage and a 72.3 minute reduction in the second stage compared to the control group, which was statistically significant (P<0.001). In comparison to the supportive care group, the mean length of the first and second stages of labor was higher in the acupressure group; however, the difference was not statistically significant (P>0.005). Lee et al. compared labor length in acupressure and palpation groups (Lee, Chang, & Kang, 2004). In that study, the active phase of delivery was considered from 3cm dilation to complete dilation. The study results demonstrated that the length of the active phase of delivery was significantly lower in the experimental group compared to the control group, which is consistent with the findings of the current study. Zeisler stated that acupuncture played a critical role in puberty, acceleration of opening of the cervix, and reduction of labor length. Therefore, due to the positive effects of this method on reduction of labor length, he introduced it as an effective method in controlling labor (Zeisler, Tempfer, Mayerhofer, Barrada, & Husslein, 1998). Reduction of labor length might have resulted from the reduction of pain and its resultant anxiety. In fact, acupressure might lead to release of endogenous opioids and decrease pain and anxiety. Reduction of delivery pain in the acupressure group can be justified by gate control theory of pain and Melzack's neuromatrix theory. Based on gate control theory of pain, acupressure activates thick nerve fibers and closes the pain gate and in this way, prevents pain transfer. According to this theory, stimulation of skin creates nervous impulses which are transferred to the spinal cord system. These impulses are either inhibited or increased in the spine. The impulses which are transferred by the thick fibers close the pain gate and, consequently, reduce pain. Moreover, stimulation of thick fibers impulses by pressure leads to more closure of the gate (Setax & Pomeranz, 2006). According to Melzack's theory, pain matrix is composed of three main nervous components, namely sensory way which passes the thalamus and sensory cortex, emotional way which passes the limbic system, and body self-recognition way which includes parietal lobe of cortex. Thus, pain can be sensory, emotional, and cognitive (Chalmers & Wolman, 1993). Hence, in the present study, acupressure affected the sensory way, prevented message transfer to the brain, and reduced the perception of pain. In contrast, Lawrence mentioned that acupuncture had no effects on reduction of labor length (Lawrence, Lewis, Hofmeyr, Dowswell, & Styles, 2009). The difference between that study and the present one might be due to different definitions of the first stage of labor. In the present study and other similar studies, the first stage of labor began from 3cm dilation to complete dilation. Lawrence, however, considered this stage from the time the number, length, and intensity of contractions were sufficient for opening of the cervix. It is noteworthy that in none of the above-mentioned studies, acupuncture or acupressure increased the first stage of labor. Moreover, not only these non-pharmacological methods did not reduce or inhibit the uterine contractions, but they also sedated the delivery pain and improved the delivery progress. The findings of the current study showed that the length of the second stage of labor was shorter among the women who received acupressure at BL32 acupoint compared to the control group. In contrast, Lee et al. conducted a clinical trial and indicated no significant difference between the acupressure and the palpation group regarding the mean length of the second stage of labor; i.e., since the complete dilation up to delivery (30.3±22.6 minutes in the acupressure group and 44.8±40.0 minutes in the palpation group). They reported that acupressure was only effective in reduction of the first stage of labor (138.6±62.0 in the acupressure group and 191.2±83.7 in the control group). The difference between Lee's study and the present one might be due to the fact that Lee applied pressure during the contractions. Thus, length of pressure application was different relative to the length of contractions. In the present study, on the other hand, application of pressure was started at the beginning of contractions with similar lengths and intervals for all the participants. The findings of the current study revealed a significant difference among the three groups regarding the first- and fifth-minute Apgar scores (P<0.001). The frequency of Apgar scores<8 in the first and fifth minutes was higher in the control group compared to the supportive care and acupressure groups. Additionally, the frequency of first- and fifth-minute Apgar scores≥8 was higher in the supportive care and acupressure groups in comparison to the control group. After 50 years, Apgar scoring system is still the best method for evaluation of newborn infants' prognosis (Casey, McIntire, & Leveno, 2001). Apgar score has been proved to be the best standard method for evaluation of infants' health immediately after birth (Jepson, Talashek, & Tichy, 1991). First-minute Apgar score indicated the newborn infants' need for resuscitation. Besides, fifth-minute Apgar score determines the probability of death or nervous complications more precisely (Roland, 1987). Apgar score, in fact, predicts infants' chance of survival (Casey et al., 2001). Cunningham et al. demonstrated that prolonged labor was accompanied by Apgar scores<7 due to long labor stages and disruption of delivery phases (Cunningham, Leveno, Bloom, Hauth, Rouse, & Spong, 2010). Furthermore, several researchers have shown that increase in the length of the second stage of labor endangered both maternal and fetal health (Saunders, Paterson, & Wadsworth, 1992) and increased the risk of complications as well as prenatal mortality (Piper, Bolling, & Newton, 1991). In the study performed by Flan, the mean of first-minute Apgar scores was 8.8 (7-10) in the active group and 7.5 (1-10) in the resting group. Besides, the two groups' means of fifth-minute Apgar scores were 9.9 (9-10) and 9.4 (7-10), respectively (Flynn & Kelly, 1987). In the same line, Hemminki (Hemminki & Lenck, 1985), Andrews (Andrews & Chrzanowski, 1990), and Ben et al. (2010) revealed that mother's activity during delivery pain reduced the length of labor stages, improved maternal and fetal outcomes, improved infants' first- and fifth-minute Apgar scores, and reduced the rate of transfer to the neonatal ward. These results were all in agreement with those of the present study. However, Liu (1989) and Stewart (Stewart & Calder, 1984) reported that mother's activity had no effects on improvement of infants' Apgar scores. The difference between these two studies and the current one might result from the fact that they only investigated physically supporting the mother. In the present study, however, the doula provided the mother with physical support (suggestion of appropriate positions and activities) as well as emotional and mental support which reduced mothers' anxiety, improved her self-confidence, and decreased labor disorders. BODY.5. CONCLUSION: The findings of the present study showed that supportive care and acupressure reduced the length of labor and increased the infants' Apgar scores compared to the control group. Therefore, these two non-pharmacological methods which are easy to perform and are not accompanied by any side effects can be employed during labor to achieve better delivery outcomes.

TITLE: Ticagrelor vs. clopidogrel in patients with non-ST-elevation acute coronary syndrome with or without revascularization: results from the PLATO trial ABSTRACT.AIMS: The optimal platelet inhibition strategy for ACS patients managed without revascularization is unknown. We aimed to evaluate efficacy and safety of ticagrelor vs. clopidogrel in the non-ST-elevation acute coronary syndrome (NSTE-ACS) subgroup of the PLATO trial, in the total cohort, and in the subgroups managed with and without revascularization within 10 days of randomization. ABSTRACT.METHODS AND RESULTS: We performed a retrospective analysis of the primary endpoint of cardiovascular death/myocardial infarction/stroke. Among 18 624 PLATO patients, 11 080 (59%) were categorized as NSTE-ACS at randomization. During the initial 10 days, 74% had angiography, 46% PCI, and 5% CABG. In NSTE-ACS patients, the primary endpoint was reduced with ticagrelor vs. clopidogrel [10.0 vs. 12.3%; hazard ratio (HR) 0.83; 95% confidence interval (CI) = 0.74–0.93], as was myocardial infarction (6.6 vs. 7.7%; HR 0.86; 95% CI = 0.74–0.99), cardiovascular death (3.7 vs. 4.9%; HR 0.77; 95% CI = 0.64–0.93), and all-cause death (4.3 vs. 5.8%; HR 0.76; 95% CI = 0.64–0.90). Major bleeding rate was similar between treatment groups (13.4 vs. 12.6%; HR 1.07; 95% CI = 0.95–1.19), but ticagrelor was associated with an increase in non-CABG major bleeding (4.8 vs. 3.8%; HR 1.28; 95% CI = 1.05–1.56). Within the first 10 days, 5366 (48.4%) patients were managed without revascularization. Regardless of revascularization or not, ticagrelor consistently reduced the primary outcome (HR 0.86 vs. 0.85, interaction P = 0.93), and all-cause death (HR 0.75 vs. 0.73, interaction P = 0.89) with no significant increase in overall major bleeding. ABSTRACT.CONCLUSION: In patients with NSTE-ACS, benefit of ticagrelor over clopidogrel in reducing ischaemic events and total mortality was consistent with the overall PLATO trial, independent of actually performed revascularization during the initial 10 days. BODY: See page 2055 for the editorial comment on this article (doi:10.1093/eurheartj/ehu202) BODY.INTRODUCTION: Dual antiplatelet therapy with aspirin and a P2Y12 inhibitor is standard of care in acute coronary syndromes (ACS).1,2 Ticagrelor is the first reversibly binding direct P2Y12 inhibitor. As opposed to clopidogrel and prasugrel, it does not require enzymatic activation, and causes faster, greater, and more consistent platelet inhibition compared with clopidogrel.3,4 The Platelet Inhibition and Patient Outcomes (PLATO) trial compared ticagrelor with clopidogrel in patients with ACS. Ticagrelor was superior in preventing ischaemic events (the composite of death from vascular causes, myocardial infarction, and stroke), as well as death from any cause, without a significant increase in all-cause major bleeding. PLATO included both invasively and non-invasively managed patients. The decision on which management strategy to pursue was made by the investigator.5 The third-generation thienopyridine prasugrel is, like clopidogrel, an irreversible P2Y12 inhibitor, but with faster and more consistent platelet inhibition.6 Prasugrel, when compared with clopidogrel, reduced ischaemic events in patients with ACS planned for PCI in the Trial to Assess Improvement in Therapeutic Outcomes by Optimizing Platelet Inhibition with Prasugrel (TRITON),7 but in non-ST-elevation acute coronary syndrome (NSTE-ACS) patients planned for management without revascularization, prasugrel showed no benefit over clopidogrel in the Targeted Platelet Inhibition to Clarify the Optimal Strategy to Medically Manage Acute Coronary Syndromes (TRILOGY-ACS) trial.8 The aim of the present study was to explore the effect of ticagrelor vs. clopidogrel in the total NSTE-ACS subgroup of the PLATO trial and also stratified by initial management with or without revascularization. BODY.METHODS.STUDY DESIGN: The PLATO trial (ClinicalTrials.gov No. NCT00391872) was an international randomized double-blind, double-dummy phase III study comparing ticagrelor with clopidogrel in ACS. Details of the study design and overall results have been published previously.5,9 Briefly, a total of 18 624 patients were randomized to receive either ticagrelor or clopidogrel on background treatment with aspirin. The study included patients with ST-elevation MI intended for primary PCI, as well as patients with non-ST-elevation ACS, regardless if aimed for an initial invasive or non-invasive treatment strategy. It adhered to the Declaration of Helsinki, to the specifications of the International Conference of Harmonization, and to Good Clinical Practice; and was approved by national and institutional regulatory authorities and ethics committees. All participants provided written informed consent. BODY.METHODS.PATIENTS: Two or more of the following inclusion criteria were required for patients enrolled without ST-elevation ACS at admission: (1) ST-segment changes on ECG indicating ischaemia [ST-segment depression or transient elevation (≥1 mm) in at least two contiguous leads]; (2) positive biomarker indicating myocardial necrosis (troponin I or T or CK-MB above the upper limit of normal); (3) one of the following: ≥60 years of age, previous myocardial infarction or coronary artery bypass surgery, coronary artery disease with ≥50% stenosis in ≥2 vessels, previous ischaemic stroke, transient ischaemic attack (TIA), carotid stenosis, cerebral revascularization, diabetes mellitus, peripheral artery disease, or chronic renal dysfunction. In the current analysis, NSTE-ACS was in the data-base defined as absence of either persistent ST-segment elevation of 1 mV for 20 min in two contiguous leads or new (or presumed new) left bundle branch block in entry ECG. In addition to the overall NSTE-ACS population, we also analysed outcomes in the subgroups of NSTE-ACS patients who initially underwent revascularization and those who were treated without early revascularization. Both cohorts required endpoint-free survival for 10 days post-randomization to determine revascularization status. The no-revascularization subgroup was defined as NSTE-ACS patients who did not undergo any revascularization procedure (PCI or CABG) with or without angiography during the first 10 days. BODY.METHODS.ENDPOINTS: The primary efficacy endpoint of the present study was the composite of cardiovascular death, myocardial infarction, (excluding silent infarctions) and stroke. Each component alone and all-cause death were secondary efficacy endpoints. Major bleeding by PLATO criteria9 and life-threatening/fatal bleeding were the primary safety endpoints. Secondary safety endpoints were CABG-related major bleeding, non-CABG-related major bleeding, minor bleeding, intracranial bleeding, and fatal bleeding. Additional secondary safety outcomes were bleeding events as defined by TIMI (major, minor, and non-CABG related major) and GUSTO (severe and moderate). An independent central adjudication committee, unaware of treatment assignments, assessed all endpoints. Major bleeding as defined by TIMI was recorded from the electronic case report form, where a drop of 50 g/L in haemoglobin was used as a cut-off, but this did not necessarily require clinical evidence of bleeding. BODY.METHODS.STATISTICAL ANALYSES: Patient characteristics and medical history, in-hospital procedures and medications, discharge ACS status, and post-discharge medications are presented by treatment in the NSTE-ACS patient cohort as well as by revascularization status subgroup. Continuous variables are presented as median and 25th–75th percentiles; categorical variables are presented as number and percentage. The treatment effect of ticagrelor vs. clopidogrel on the primary and secondary endpoints was compared within the subgroup of NSTE-ACS patients. Kaplan–Meier estimated event rates at 360 days and total number of observed events during the study were presented for each endpoint. Hazard ratios (HR), confidence intervals (CI), and P-values from unadjusted Cox proportional hazards regression models were presented. Kaplan–Meier estimated event rates were plotted by treatment for the primary efficacy endpoint, all-cause death, major bleeding, and non-CABG related major bleeding. To examine whether the effect of ticagrelor in patients with NSTE-ACS differed based on early revascularization, Cox proportional hazards models were fitted for each endpoint using a treatment-by-revascularization interaction term. In these models, we adjusted for region of the world to account for differences in revascularization practice and the time-to-event was measured from a landmark at 10 days post-randomization. Kaplan–Meier rates 350 days post-landmark are presented for each treatment/revascularization category along with HR for ticagrelor vs. clopidogrel in the revascularization and medical management subgroups. Interaction P-values assess whether the treatment effect is different depending on revascularization status. Kaplan–Meier event rates adjusted for region are plotted by the four treatment/revascularization categories for the primary efficacy endpoint, all-cause death, major bleeding, and non-CABG related major bleeding. For consistency with the PLATO design paper,9 we also performed a 30-day landmark analysis; and as a sensitivity analysis, we also performed a 10-day landmark analysis of the full study population. Finally, the association of treatment with the primary efficacy endpoint was evaluated within several patient cohorts using Cox proportional hazards models, separately within the revascularization and non-revascularization subgroups. Forest plots present the HR for ticagrelor vs. clopidogrel in each subgroup cohort. Endpoints are defined using intent to treat and a P-value of 0.05 is used to denote statistical significance. Data were analysed using SAS version 9.2 for all analyses. BODY.RESULTS.PATIENT CHARACTERISTICS: The PLATO trial included 18 624 patients with ACS, of which 11 080 patients were classified as NSTE-ACS at randomization. Of these, 5581 were randomized to ticagrelor and 5499 to clopidogrel (Figure 1). The baseline and in-hospital characteristics were similar between groups in the overall NSTE-ACS population (Table 1). In the overall NSTE-ACS group, 74% of patients had a coronary angiography performed, 46% underwent PCI, and 5% CABG. The discharge diagnosis (in this population categorized as NSTE-ACS at admission) was NSTEMI in 65%, STEMI in 8%, and unstable angina/other in 27%. Table 1Baseline characteristics and invasive procedures by randomized treatment in the overall NSTE-ACS population, and by initial treatment strategy (within the first 10 days after randomization) Overall NSTE-ACSNSTE-ACS with revascularizationNSTE-ACS without revascularizationTicagrelor (N = 5581)Clopidogrel (N = 5499)Ticagrelor (N = 2873)Clopidogrel (N = 2841)Ticagrelor (N = 2708)Clopidogrel (N = 2658)Demographics Age, median (25th–75th percentile), years64 (56–72)64 (56–72)63 (55–71)63 (55–71)65 (57–73)65 (57–73) Age ≥ 75 years, n (%)955 (17.1)1024 (18.6)420 (14.6)460 (16.2)535 (19.8)564 (21.2) Female gender, n (%)1746 (31.3)1746 (31.8)706 (24.6)716 (25.2)1040 (38.4)1030 (38.8) Body weight <60 kg, n (%)398 (7.2)389 (7.1)165 (5.8)172 (6.1)233 (8.6)217 (8.2) Body mass index, median (25th–75th percentile), kg/m227.5 (24.8–30.8)27.4 (24.8–30.5)27.5 (24.9–30.8)27.5 (24.9–30.5)27.5 (24.7–30.6)27.3 (24.6–30.5) GRACE risk score nomogram130 (112–150)130 (112–149)128 (110–145)127 (110–145)133 (114–154)134 (116–153)Cardiovascular risk factors, n (%) Current smoking1636 (29.4)1640 (29.9)1000 (34.8)960 (33.8)636 (23.6)680 (25.6) Hypertension3915 (70.2)3835 (69.8)1881 (65.5)1886 (66.4)2034 (75.3)1949 (73.4) Dyslipidaemia2885 (51.8)2852 (51.9)1515 (52.8)1569 (55.2)1370 (50.7)1283 (48.3) Diabetes mellitus1608 (28.9)1520 (27.7)756 (26.3)747 (26.3)852 (31.6)773 (29.1)Medical history, n (%) Angina pectoris2932 (52.6)2890 (52.6)1337 (46.5)1356 (47.7)1595 (59.1)1534 (57.8) Myocardial infarction1400 (25.1)1410 (25.7)609 (21.2)608 (21.4)791 (29.3)802 (30.2) Congestive heart failure397 (7.1)429 (7.8)99 (3.4)103 (3.6)298 (11.0)326 (12.3) Percutaneous coronary intervention (PCI)944 (16.9)918 (16.7)537 (18.7)517 (18.2)407 (15.1)401 (15.1) Coronary artery bypass graft (CABG)434 (7.8)474 (8.6)214 (7.4)218 (7.7)220 (8.1)256 (9.6) Transient ischaemic attack185 (3.3)189 (3.4)82 (2.9)74 (2.6)103 (3.8)115 (4.3) Non-hemorrhagic stroke246 (4.4)243 (4.4)81 (2.8)98 (3.5)165 (6.1)145 (5.5) Peripheral arterial disease400 (7.2)413 (7.5)174 (6.1)204 (7.2)226 (8.4)209 (7.9) Chronic renal disease273 (4.9)279 (5.1)117 (4.1)108 (3.8)156 (5.8)171 (6.4)Physical findings, median (25th–75th percentile) Heart rate (bpm)72 (64–80)72 (64–81)71 (62–80)72 (63–80)72 (64–82)72 (64–82) Systolic blood pressure (mmHg)135 (120–150)134 (120–150)135 (120–150)135 (120–150)134 (120–150)132 (120–150) Diastolic blood pressure (mmHg)80 (70–89)80 (70–87)80 (70–89)80 (70–87)80 (70–89)80 (70–87)Risk indicators, n (%) Troponin positive, n (%)4356 (80.8)4323 (81.3)2522 (89.6)2486 (89.3)1834 (71.3)1837 (72.4) ST depression (≥1 mm)3158 (56.8)3201 (58.4)1547 (54.0)1535 (54.2)1611 (59.8)1666 (62.8) TIMI risk score > 24838 (89.7)4785 (89.8)2584 (91.3)2566 (91.8)2254 (88.0)2219 (87.7)Type of ACS at discharge, n (%) STEMI449 (8.1)437 (8.0)330 (11.5)305 (10.7)119 (4.4)132 (5.0) NSTEMI3605 (64.8)3525 (64.3)2045 (71.2)2025 (71.3)1560 (58.0)1500 (56.7) UA/other1509 (27.1)1524 (27.8)497 (17.3)510 (18.0)1012 (37.6)1014 (38.3)Antithrombotic treatment during index hospitalization, n (%) Aspirin5386 (96.6)5316 (96.8)2797 (97.4)2779 (97.8)2589 (95.9)2537 (95.8) Unfractionated heparin2910 (52.1)2856 (51.9)1845 (64.2)1845 (64.9)1065 (39.3)1011 (38.0) Low molecular weight heparin3181 (57.0)3084 (56.1)1568 (54.6)1501 (52.8)1613 (59.6)1583 (59.6) Fondaparinux194 (3.5)191 (3.5)87 (3.0)76 (2.7)107 (4.0)115 (4.3) Bivalirudin140 (2.5)133 (2.4)135 (4.7)122 (4.3)5 (0.2)11 (0.4) GP IIb/IIIa inhibitor1170 (21.0)1117 (20.3)982 (34.2)963 (33.9)188 (6.9)154 (5.8)Other drug from randomization to end of study, n (%) Beta-blocker4768 (85.4)4685 (85.2)2536 (88.3)2512 (88.4)2232 (82.4)2173 (81.8) ACE-inhibitor and/or ARB4693 (84.2)4602 (83.8)2447 (85.2)2433 (85.6)2246 (83.2)2169 (81.9) Statin5196 (93.1)5109 (92.9)2777 (96.7)2745 (96.6)2419 (89.3)2364 (88.9) Proton-pump inhibitor2776 (49.7)2613 (47.5)1618 (56.3)1536 (54.1)1158 (42.8)1077 (40.5) Calcium channel inhibitor1553 (27.8)1515 (27.6)720 (25.1)723 (25.4)833 (30.8)792 (29.8) Diuretic2393 (42.9)2280 (41.5)1073 (37.3)1037 (36.5)1320 (48.9)1243 (46.9)Invasive procedures Coronary angiography During first 10 days, n (%)4143 (74.5)4072 (74.2)2873 (100.0)2841 (100.0)1270 (47.2)1231 (46.6) After first 10 days, n (%)357 (6.4)335 (6.1)0 (0)0 (0)357 (13.3)335 (12.7) PCI During first 10 days, n (%)2590 (46.4)2550 (46.4)2590 (90.1)2550 (89.8)0 (0)0 (0) After first 10 days, n (%)279 (5.0)291 (5.3)11 (0.4)3 (0.1)268 (9.9)288 (10.8) CABG During first 10 days, n (%)296 (5.3)305 (5.5)296 (10.3)305 (10.7)0 (0)0 (0) After first 10 days, n (%)382 (6.8)375 (6.8)55 (1.9)55 (1.9)327 (12.1)320 (12.0) Figure 1Trial profile—patients classified by entry ECG and by treatment during the first 10 days after randomization. At 10 days post-randomization, 5366 patients were alive and had not undergone revascularization. There were regional differences in the proportion of patients undergoing revascularization during the initial 10 days (Asia/Australia 44%, Central/South America 40.3%, Europe/Middle East/Africa 49.8%, North America 71.6%). Non-revascularized and revascularized patients were of similar age (63 vs. 65), but patients who did not undergo revascularization were more likely to be of female gender (39 vs. 25%), and more likely to have comorbidities (e.g. previous myocardial infarction, heart failure, renal disease). Although non-revascularized patients had more comorbidities, they were less likely to be troponin I positive (Table 1). The proportion of patients with TIMI risk score >2 was slightly higher in the revascularization group (92 vs. 88%). During the first 10 days, 47% of non-revascularized patients underwent coronary angiography. When including only those who had angiography during the initial 10 days, female gender was more common in the non-revascularized (36%) than revascularized (25%). No significant coronary artery disease at angiography was noted in 32 and 0.7% of the non-revascularized and revascularized, respectively. BODY.RESULTS.EFFICACY IN THE OVERALL NON-ST-ELEVATION ACUTE CORONARY SYNDROME POPULATION: Efficacy and safety outcomes of the overall NSTE-ACS population are summarized in Table 2, and Kaplan–Meier curves are shown in Supplementary material online. The incidence of the primary composite endpoint was reduced with ticagrelor vs. clopidogrel (10.0 vs. 12.3%; HR 0.83; 95% CI = 0.74–0.93; P = 0.0013) (see Supplementary material online, Figure S1A). Cardiovascular death occurred less often in the ticagrelor group than in the clopidogrel group (3.7 vs. 4.9%; HR 0.77; 95% CI = 0.64–0.93; P = 0.0070), and myocardial infarction was also less common with ticagrelor vs. clopidogrel (6.6 vs. 7.7%; HR 0.86; 95% CI = 0.74–0.99; P = 0.0419), whereas stroke incidence did not differ significantly between treatment arms (1.3 vs. 1.4%; HR 0.95; 95% CI 0.69–1.33; P = 0.79). All-cause death was reduced in those treated with ticagrelor vs. clopidogrel (4.3 vs. 5.8%; HR 0.76; 95% CI = 0.64–0.90; P = 0.0020) (see Supplementary material online, Figure S1B). Table 2Efficacy and safety outcomes in patients with NSTE-ACSTicagrelor % (n)Clopidogrel % (n)HR (95% CI)P-valueEfficacy endpoints CV death/MI (excluding silent)/stroke10.0 (533)12.3 (630)0.83 (0.74, 0.93)0.0013 All-cause death/MI(excl. silent)/stroke10.5 (557)13.0 (664)0.82 (0.73, 0.92)0.0006 CV death/MI(all)/stroke/severe recurrent ischaemia/recurrent ischaemia/TIA/arterial thrombotic event15.5 (824)17.8 (918)0.88 (0.80, 0.96)0.0058 Myocardial infarction (excluding silent)6.6 (345)7.7 (392)0.86 (0.74, 0.99)0.0419 Cardiovascular death (includes vascular and unknown deaths)3.7 (194)4.9 (247)0.77 (0.64, 0.93)0.0070 Stroke1.3 (69)1.4 (71)0.95 (0.69, 1.33)0.79 All-cause death4.3 (224)5.8 (290)0.76 (0.64, 0.90)0.0020Safety endpoints Major bleeding (study criteria)13.4 (660)12.6 (618)1.07 (0.95, 1.19)0.26 Major or minor bleeding (study criteria)18.2 (900)16.3 (794)1.14 (1.03, 1.25)0.0078 Non-CABG related major bleeding (study criteria)4.8 (225)3.8 (176)1.28 (1.05, 1.56)0.0139 Fatal bleeding0.3 (13)0.4 (18)0.72 (0.35, 1.47)0.37 Life threatening or fatal bleeding (study criteria)6.6 (331)6.5 (315)1.05 (0.90, 1.22)0.56 Intracranial bleeding0.3 (14)0.2 (7)2.01 (0.81, 4.99)0.13 Other major bleeding7.2 (344)6.6 (318)1.08 (0.93, 1.25)0.34 Major bleeding (TIMI criteria)9.2 (452)8.7 (422)1.07 (0.94, 1.22)0.33 Major or minor bleeding (TIMI criteria)13.2 (653)12.3 (602)1.08 (0.97, 1.21)0.16 Non-CABG related major bleeding (TIMI criteria)2.9 (137)2.2 (99)1.39 (1.07, 1.80)0.0131 GUSTO severe bleeding3.1 (146)3.2 (151)0.96 (0.77, 1.21)0.74 GUSTO moderate or severe bleeding8.6 (416)7.8 (382)1.08 (0.94, 1.25)0.25Each treatment group is summarized as Kaplan–Meier rates at 360 days and total number of events during the study. P-values and hazard ratios (95% CI) come from unadjusted Cox models testing ticagrelor vs. clopidogrel. BODY.RESULTS.SAFETY: With ticagrelor when compared with clopidogrel, there was no significant difference in PLATO major bleeding (13.4 vs. 12.6%; HR 1.07; 95% CI = 0.95–1.19; P = 0.26), but a higher rate of non-CABG-related major bleeding (4.8 vs. 3.8%; HR 1.28; 95% CI = 1.05–1.56; P = 0.0139) (see Supplementary material online, Figure S2A and B). There was no significant difference in the rate of life-threatening or fatal bleeding (6.6 vs. 6.5%; HR 1.05; 95% CI = 0.90–1.22, P = 0.56), nor any significant difference in the rate of intracranial bleeding with ticagrelor compared with clopidogrel (0.3 vs. 0.2%; HR 2.01; 95% CI = 0.81–4.99; P = 0.13). The composite of major or minor bleeding (by PLATO criteria) occurred more often in the ticagrelor group (18.2 vs. 16.3%; HR 1.14; 95% CI = 1.03–1.25, P = 0.0078). When assessed by TIMI criteria, there was no significant difference in major or minor bleeding (13.2 vs. 12.3%; HR 1.08; 95% CI = 0.97–1.21; P = 0.16). TIMI major bleeding, GUSTO severe, and GUSTO moderate or severe bleeding also did not appear to differ significantly between ticagrelor and clopidogrel, whereas TIMI non-CABG-related major bleeding was more common in the ticagrelor group (Table 2). BODY.RESULTS.EFFICACY AND SAFETY ACCORDING TO TREATMENT STRATEGY: Event rates were considerably higher in NSTE-ACS patients treated without revascularization compared with patients undergoing revascularization during the initial 10 days. For both revascularized and non-revascularized patients, there were similar proportional reductions of the primary endpoint with ticagrelor compared with clopidogrel (HR 0.86 vs. 0.85, interaction P = 0.93) (Figure 2A, Table 3) consistent with the overall trial. There was also a consistent reduction in all-cause death (HR 0.75 vs. 0.73; interaction P = 0.89) (Figure 2B). No significant difference in overall major bleeding was seen with ticagrelor vs. clopidogrel within each treatment strategy (revascularization/no revascularization) (Figure 3A). There was a higher incidence of non-CABG-related major bleeding with ticagrelor vs. clopidogrel in patients with NSTE-ACS with no significant interaction by invasive treatment strategy (HR 1.32 vs. 1.07; interaction P = 0.43) (Figure 3B). The primary outcome was reduced in major subgroups in both revascularized and non-revascularized patients (see Supplementary material online, Figures S3 and S4). The results were consistent with a 30-day landmark for revascularization (see Supplementary material online, Table S1), as well as in the full study population (see Supplementary material online, Table S2). In patients who underwent angiography during the initial 10 days with or without significant coronary disease, the effect of ticagrelor vs. clopidogrel was consistent (see Supplementary material online, Table S3). Table 3Interaction of ticagrelor treatment and revascularization within 10 days (adjusting for region) NSTE-ACS with revascularizationNSTE-ACS without revascularizationInteraction PNTicagrelor KM rateClopidogrel KM rateHR (95% CI)NTicagrelor KM rateClopidogrel KM rateHR (95% CI)Efficacy endpoints CV death/MI (excluding silent)/stroke54165.116.100.86 (0.68, 1.09)51899.6311.600.85 (0.72, 1.01)0.93 All-cause death/MI (excl. silent)/stroke54165.446.600.85 (0.67, 1.06)518910.1512.530.84 (0.71, 0.99)0.94 CV death/MI (all)/stroke/severe recurrent ischaemia/recurrent ischaemia/TIA/arterial thrombotic event52908.7310.310.86 (0.71, 1.03)510914.1415.160.97 (0.84, 1.13)0.29 Myocardial infarction (excluding silent)54383.523.880.90 (0.68, 1.21)52016.046.680.94 (0.75, 1.17)0.85 Cardiovascular death (includes vascular and unknown deaths)56481.642.330.76 (0.52, 1.13)52174.075.440.75 (0.58, 0.98)0.95 Stroke56320.670.591.18 (0.60, 2.34)52091.481.690.92 (0.58, 1.46)0.56 All-cause death56482.032.880.75 (0.53, 1.07)52174.776.650.73 (0.57, 0.93)0.89Safety endpoints Major bleeding (study criteria)49835.254.681.10 (0.84, 1.44)493111.8311.431.05 (0.88, 1.26)0.82 Major or minor bleeding (study criteria)48427.766.351.22 (0.97, 1.54)484714.5913.961.07 (0.91, 1.25)0.34 Non-CABG major bleeding (study criteria)52703.142.381.32 (0.92, 1.90)49332.782.791.07 (0.74, 1.56)0.43 Fatal or life-threatening major bleeding (study criteria)51732.252.011.18 (0.79, 1.76)49625.776.110.95 (0.75, 1.22)0.37 Other major bleeding (study criteria)51783.102.851.02 (0.72, 1.45)49456.505.681.16 (0.91, 1.49)0.55 Major bleeding (TIMI criteria)51023.422.791.21 (0.86, 1.70)49528.048.390.97 (0.79, 1.20)0.28 Major or minor bleeding (TIMI criteria)49905.244.561.12 (0.85, 1.47)493311.7811.161.08 (0.91, 1.29)0.85 Non-CABG major bleeding (TIMI criteria)53161.881.191.66 (1.01, 2.72)49522.051.841.19 (0.76, 1.87)0.34 GUSTO severe bleeding52861.161.490.75 (0.45, 1.26)49462.752.461.13 (0.77, 1.65)0.22 GUSTO moderate or severe bleeding51343.933.301.13 (0.82, 1.55)49457.185.961.19 (0.95, 1.51)0.78Kaplan–Meier (KM) rates 350 days after day 10 post-randomization. Figure 2Efficacy endpoints stratified by management strategy—Kaplan–Meier estimates of time to first occurrence of: (A) primary endpoint, (B) all-cause death, from 10 days post-randomization onward. Figure 3Bleeding stratified by revascularization—Kaplan–Meier estimate of (A) time to major bleeding according to the PLATO criteria from day 10 post-randomization, and (B) time to non-CABG major bleeding. BODY.DISCUSSION: In this subgroup analysis, ticagrelor compared with clopidogrel reduced the composite endpoint of cardiovascular death, myocardial infarction, and stroke as well as the individual endpoints of cardiovascular death, myocardial infarction, and all-cause death without any significant difference in major bleeding in patients with an entry diagnosis of NSTE-ACS. The event curves for the primary composite endpoint and total mortality separated continuously for the duration of the trial. The benefits with ticagrelor were observed both in patients who underwent and in those who did not undergo early revascularization, regardless whether angiography was performed or not. These results are consistent with the previously reported subgroup analysis of all ACS patients with an intended strategy (prior to randomization) of no revascularization, where about one-fifth still underwent PCI and about 4% had CABG surgery before discharge.10 Although current guidelines advocate early invasive management in NSTE-ACS,1,2 a large proportion of patients are managed non-invasively.11,12 Patients who are managed without revascularization usually have more comorbidities, higher risk of bleeding, and inferior outcome than patients who are revascularized.13 The optimal platelet inhibition strategy in these patients has been uncertain. P2Y12 inhibition has previously been shown to reduce ischaemic events in NSTE-ACS patients managed without revascularization. In the Clopidogrel in Unstable Angina to Prevent Recurrent Events (CURE) trial, which randomized NSTE-ACS patients to receive either clopidogrel or placebo (on background aspirin treatment), 64% of patients did not undergo revascularization after randomization. There were almost identical relative reductions in CV death/MI/stroke with clopidogrel in the non-invasive and invasive subgroups.14 The Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management and Avoidance (CHARISMA) trial studied the addition of clopidogrel to aspirin in a stable, more heterogeneous population at risk for atherothrombotic events. Overall, the combination was not more effective than aspirin monotherapy, but a trend towards benefit was noted in those with symptomatic atherosclerotic disease, whereas a trend towards harm was seen in those included based only on multiple cardiovascular risk factors.15 This issue was also recently studied in the TRILOGY-ACS trial, in which NSTE-ACS patients intended for management without revascularization were prospectively randomized to receive either prasugrel or clopidogrel (both irreversible P2Y12 inhibitors). The results showed no significant overall benefit of prasugrel over clopidogrel during 24 months, even though the Kaplan–Meier curves for the efficacy endpoints tended to separate after 1 year,8 and with a reduction of the primary outcome in the subgroup who underwent angiography.16 In the no revascularization subgroup of the current PLATO NSTE-ACS substudy, there were consistent benefits with ticagrelor when compared with clopidogrel concerning both mortality and non-fatal ischaemic events. Although the no revascularization subgroup of the present PLATO substudy seems similar to the TRILOGY-ACS population, it is impossible to compare the effect of the respective new P2Y12 inhibitor vs. clopidogrel across the two studies. Patients with intention for treatment without revascularization were prospectively studied in TRILOGY-ACS, while the present PLATO substudy was a post hoc stratification with subgroups of revascularization/no revascularization defined post randomization and post procedures. The TRILOGY-ACS population also was of higher risk with more prevalent comorbidities such as hypertension, dyslipidaemia, and diabetes, and had higher rates of previous myocardial infarction and revascularization procedures. In ACS patients managed with revascularization, more potent P2Y12 inhibition has been associated with better outcomes, as shown in the intention for invasive management subgroup analysis of PLATO for ticagrelor17 and in TRITON for prasugrel.7 In the Clopidogrel and Aspirin Optimal Dose Usage to Reduce Recurrent Events–Seventh Organization to Assess Strategies in Ischemic Syndromes (CURRENT OASIS-7) trial also, intensified P2Y12 inhibition with double dose clopidogrel for the first 7 days showed no significant difference in outcome in the overall population,18 but a reduction in cardiovascular events, including stent thrombosis in the pre-specified (albeit post-randomization) subgroup undergoing PCI.19 With more potent platelet inhibition, bleeding complications have usually been increasing. In TRITON-TIMI 38, where prasugrel vs. clopidogrel was evaluated in ACS, prasugrel demonstrated a reduction in thrombotic events, but at the cost of significantly increased rates of major bleeding, including fatal bleeding, particularly in patients at high bleeding risk defined as high age and low body weight.7 In TRILOGY-ACS on the other hand, which included a lower maintenance dose, prasugrel did not cause any increased major bleeding rate, including patients >75 years of age. In the present analysis, there was no significant difference in PLATO-defined total major bleeding with ticagrelor compared with clopidogrel. However, the incidence of non-CABG-related major bleeding was significantly higher in the ticagrelor group. There was no significant difference in life-threatening or fatal bleeding or major bleeding as defined by the TIMI criteria, but the composite of PLATO major/minor bleeding was increased with ticagrelor. BODY.DISCUSSION.STUDY LIMITATIONS: There are several limitations to this work. The sample size is large, which gives high power to detect even relatively small differences in effect that may or may not be clinically important. The revascularization/no revascularization analyses were post hoc investigations of subgroups identified post-randomization, which makes the analyses subject to potential bias. Because landmark analyses were used, the risk of time-dependent confounding is acknowledged. As sensitivity analyses, we also performed landmark analyses at 30 days instead of 10 days, with consistent results. Nevertheless, the findings reported with regard to revascularization status should be interpreted strictly as exploratory and hypothesis generating. The present results based on actual revascularization strategy support and complement those of our previous analysis based on pre-randomization intention to treat with invasive or conservative management, and the results based on performed revascularization or not are consistent with the overall NSTE-ACS results, as well as the overall PLATO results. BODY.CONCLUSIONS: In this substudy of the PLATO trial, ticagrelor compared with clopidogrel consistently reduced the rates of ischaemic events and mortality without any difference in overall major bleeding in patients with an entry diagnosis of NSTE-ACS, and this effect was independent of whether or not early revascularization was performed. These results harmonize with the European Society of Cardiology (ESC) NSTE-ACS guidelines, which recommend ticagrelor in all patients at moderate-to-high risk of ischaemic events, regardless of initial treatment strategy.1 BODY.SUPPLEMENTARY MATERIAL: Supplementary material is available at European Heart Journal online. BODY.FUNDING: This work was supported by AstraZeneca, who funded the PLATO trial. Funding to pay the Open Access publication charges for this article was provided by Astra Zeneca. Conflicts of interest: D.L.: Institutional research grant from AstraZeneca to perform this research; and lecture fees from AstraZeneca. C.V.: research grant from AstraZeneca to perform this research; member of the Speakers' Bureaus for AstraZeneca, Eli Lilly & Company, and The Medicines Company. C.P.C.: research grants/support from Accumetrics, AstraZeneca, CSL Behring, Essentialis, GlaxoSmithKline, Merck, Regeneron, Sanofi, and Takeda; on advisory boards for Alnylam, Bristol-Myers Squibb, Lipimedix, and Pfizer (funds donated to charity); and holds equity in Automedics Medical Systems. R.A.H.: consulting/advisory board fees from Bristol-Myers Squibb, Sanofi, Portola Pharmaceuticals, Johnson & Johnson and Merck; grant support from Eli Lilly/Daiichi Sankyo., Merck, Portola Pharmaceuticals, Sanofi, Johnson & Johnson, Bristol-Myers Squibb, The Medicines Company, and AstraZeneca. A.H.: reports being an employee of AstraZeneca. J.M.: reports being an employee of AstraZeneca and having equity ownership. S.H.: advisory board member for AstraZeneca, Bristol-Myers Squibb, Pfizer, and Bayer; research support from GlaxoSmithKline, Pfizer, and Sanofi-Aventis. P.G.S.: research grant (to INSERM U698): NYU School of Medicine, Sanofi, Servier. Speaking or consulting: Amarin, AstraZeneca, Bayer, Boehringer-Ingelheim, Bristol-Myers-Squibb, Daiichi-Sankyo, GSK, Lilly, Medtronic, Otsuka, Pfizer, Roche, sanofi, Servier, The Medicines Company, Vivus. Stockholding: Aterovax. J.H.C.: advisory board fees from BMS, AstraZeneca, Eli Lilly/Daiichi Sankyo; consultancy fees from Merck and Servier. R.F.S.: research grants from AstraZeneca, Eli Lilly/Daiichi Sankyo, and Merck; research support from Accumetrics; honoraria from AstraZeneca, Eli Lilly/Daiichi Sankyo, Merck, Iroko, Accumetrics, and Medscape; consultancy fees from AstraZeneca, Merck, Novartis, Accumetrics, Sanofi-Aventis/Regeneron, Bristol-Myers Squibb, Eisai, Roche and Daiichi Sankyo. S.R.S.: reports no conflict of interest. L.W.: research grants from AstraZeneca, Merck & Co, Boehringer-Ingelheim, Bristol-Myers Squibb/Pfizer, GlaxoSmithKline; consultant for Merck & Co, Regado Biosciences, Evolva, Portola, C.S.L. Behring, Athera Biotechnologies, Boehringer-Ingelheim, AstraZeneca, GlaxoSmithKline, and Bristol-Myers Squibb/Pfizer; lecture fees from AstraZeneca, Boehringer-Ingelheim, Bristol-Myers Squibb/Pfizer, GlaxoSmithKline, and Merck & Co.; honoraria from Boehringer Ingelheim, AstraZeneca, Bristol-Myers Squibb/Pfizer, GlaxoSmithKline, and Merck & Co.; travel support from AstraZeneca and Bristol-Myers Squibb/Pfizer. S.K.J.: research grant from AstraZeneca, Eli Lilly, Bristol-Myers Squibb, Terumo Inc, Medtronic, and Vascular Solutions; honoraria from The Medicines Company, AstraZeneca, Eli Lilly, Bristol-Myers Squibb, and IROKO; consultant/advisory board from AstraZeneca, Eli Lilly, Merck, Medtronic, and Sanofi. BODY.SUPPLEMENTARY MATERIAL: Supplementary Data

TITLE: A Comparison of Two Delivery Modalities of a Mobile Phone-Based Assessment for Serious Mental Illness: Native Smartphone Application vs Text-Messaging Only Implementations ABSTRACT.BACKGROUND: Mobile phone–based assessment may represent a cost-effective and clinically effective method of monitoring psychotic symptoms in real-time. There are several software options, including the use of native smartphone applications and text messages (short message service, SMS). Little is known about the strengths and limitations of these two approaches in monitoring symptoms in individuals with serious mental illness. ABSTRACT.OBJECTIVE: The objective of this study was to compare two different delivery modalities of the same diagnostic assessment for individuals with non-affective psychosis—a native smartphone application employing a graphical, touch user interface against an SMS text-only implementation. The overall hypothesis of the study was that patient participants with sewrious mental illness would find both delivery modalities feasible and acceptable to use, measured by the quantitative post-assessment feedback questionnaire scores, the number of data points completed, and the time taken to complete the assessment. It was also predicted that a native smartphone application would (1) yield a greater number of data points, (2) take less time, and (3) be more positively appraised by patient participant users than the text-based system. ABSTRACT.METHODS: A randomized repeated measures crossover design was employed. Participants with currently treated Diagnostic and Statistical Manual (Fourth Edition) schizophrenia or related disorders (n=24) were randomly allocated to completing 6 days of assessment (four sets of questions per day) with a native smartphone application or the SMS text-only implementation. There was then a 1-week break before completing a further 6 days with the alternative delivery modality. Quantitative feedback questionnaires were administered at the end of each period of sampling. ABSTRACT.RESULTS: A greater proportion of data points were completed with the native smartphone application in comparison to the SMS text-only implementation (β = -.25, SE=.11, P=.02), which also took significantly less time to complete (β =.78, SE= .09, P<.001). Although there were no significant differences in participants' quantitative feedback for the two delivery modalities, most participants reported preferring the native smartphone application (67%; n=16) and found it easier to use (71%; n=16). 33% of participants reported that they would be willing to complete mobile phone assessment for 5 weeks or longer. ABSTRACT.CONCLUSIONS: Native smartphone applications and SMS text are both valuable methods of delivering real-time assessment in individuals with schizophrenia. However, a more streamlined graphical user interface may lead to better compliance and shorter entry times. Further research is needed to test the efficacy of this technology within clinical services, to assess validity over longer periods of time and when delivered on patients' own phones. BODY.INTRODUCTION: Schizophrenia is a major public health problem affecting approximately 1% of the general population. It represents both a substantive emotional burden for those involved and a socioeconomic burden in the United Kingdom [1]. There is now increasing emphasis on treating patients within the community [2]. However, symptoms, mood, and functioning can change suddenly, potentially leading to relapse, unscheduled acute care, or self-injury. Monitoring of psychotic symptoms usually relies on interview assessments infrequently conducted by clinical staff, with limited resources, reducing the capacity to detect sudden change. Additionally, interviews relying on retrospective accounts introduces bias and averaging, thereby losing clinical information [3]. A more sensitive approach would be to closely monitor patients during the course of their everyday lives. Real-time assessment of psychotic symptoms is attractive in that it can lead to early and immediate intervention, potentially preventing the deterioration of an individual's mental state. Over the past decade, assessment technologies have been increasingly employed in the monitoring of psychosis [4]. Typically, patients complete self-report questions at several different times of the day in real-world settings [5]. Early research in this field focused on the use of Personal Digital Assistants (PDAs) [6,7], but these technologies are becoming increasingly obsolescent and now occupy a very small share of the commercial market. Mobile phones have the advantage that they allow the automatic and wireless uploading of information to a central computer. As mobile phone technology becomes increasingly widespread, software applications can operate from users' own phones, omitting the need for them to carry an additional device. In a recent study, we observed that 83% (n=30/36) of a sample with psychosis currently owned a mobile phone, suggesting that usage in individuals with psychosis is comparable to the general population in the United Kingdom. Other studies have also reported high levels of familiarity with and access to mobile phone technology in individuals with serious mental illness [8]. There are multiple ways in which mobile phone-based assessment can be employed in health care. Text messages have the advantage that they are relatively easy and inexpensive to deliver and are not limited by the model or make of an individual's phone [9]. Individuals will likely be familiar with sending and receiving text messages. To date, two studies have used text messaging in the clinical care of psychotic illness. Španiel and colleagues [10] used weekly text-based assessments in order to alert consulting psychiatrists to early signs of relapse in patients with psychosis, which significantly reduced the number of inpatient admissions over one year. Additionally, Granholm and colleagues [11] have used text messages to promote cognitive behavioral therapy techniques and health-promoting behaviors. Pilot data showed a significant increase in social interaction and a reduction in hallucinations, but no significant effect on medication use. Native software applications, with graphical user interfaces, uploaded onto smartphone devices pose another viable option for conducting real-time assessment. For the purpose of this research, a smartphone will be defined as having (1) computing power, (2) a touch screen, (3) third party application development and distribution, and (4) the option of 3G connectivity and high-speed data transfer. Native smartphone applications can be purpose built allowing for greater flexibility and ease of use [9] and are now increasingly being used in the treatment of physical health problems (eg, diabetes) [12]. Our research group has recently developed one such software application for use on Android smartphones. This software enables users to respond to self-report questions relating to their symptoms on touch-screen analogue scales. Initial piloting of this technology showed little dropout and high levels of data points completed in 44 individuals experiencing psychosis, sampled for 1 week. 13 self-assessment scales representing different dimensions of psychotic illness were validated against commonly employed, gold-standard clinical interviews. However, further information is required to assess the benefits and limitations of native smartphone applications in comparison to other methods of mobile phone-based data collection. The objective of this study was to compare two different delivery modalities of the same diagnostic assessment for individuals with nonaffective psychosis—a native smartphone application employing a graphical, touch user interface against an short message service (SMS) text-only implementation. It was thought that both approaches would have certain advantages in the real-time assessment of psychosis but that the native smartphone application would display greater usability and functionality. Understanding the appropriate technology, user interface, procedures, and methods for administering mobile phone–based assessment is an important step in making it both cost- and clinically effective, with the eventual aim of integrating it into long-term illness management. The overall hypothesis of the study was that patient participants with serious mental illness would find both the smartphone application and SMS text-only implementation feasible and acceptable to use, measured by quantitative postassessment feedback questionnaire scores, the number of data points completed, and the time taken to complete the assessment. We also had three more specific predictions: (1) that the participants would complete a significantly greater number of entries on a native smartphone application when compared to a SMS text-only implementation, (2) that entries would take significantly longer to complete on a SMS text-only implementation, and (3) that the smartphone application would be appraised more favorably in quantitative feedback scores. We also asked participants about the maximum length of time that individuals thought they would be willing to complete assessment over for each delivery modality. BODY.METHODS.PARTICIPANTS: 24 community-based patients meeting or having met the criteria for a Diagnostic and Statistical Manual (Fourth Edition) diagnosis of schizophrenia (n=22) or schizoaffective disorder (n=2) as made by the clinical service and checked against DSM-IV criteria by the researcher were admitted to the study. All participants were aged 18-50 and provided informed consent to take part. Participants were required to own and have access to a mobile phone with which to use the SMS text-only implementation. Organic or substance induced psychosis were exclusion criteria. The sample was predominantly male (79%; n=19) and white British (71%; n=17) with a mean age of 33.0 (SD 9.5, min=18, max=49). Patients were recruited through Community Mental Health Teams (63%; n=15), Early Intervention Services (33%; n=8) and through a supported-living organization (4%; n=1). The mean number of past hospital admissions in the sample was 2.3 (SD 2.5, min = 0, max = 9) and the majority of the sample were currently taking antipsychotic medication (n=20). The Positive and Negative Syndrome Scale (PANSS [13]) is taken as a gold standard-clinical measure commonly used to assess psychosis. PANSS scores in this sample (at baseline) varied considerably between individuals, suggesting a range of symptom severities (positive subscale: mean 15.0 (SD 4.5, min = 7, max = 28); negative subscale: mean 12.3 (SD 3.5, min = 7, max = 21); general subscale: mean 28.8 (SD 6.8, min = 18, max = 50). BODY.METHODS.EQUIPMENT: The two different modalities for delivering real-time assessment, namely a smartphone application and an SMS text-only implementation, were designed such that protocol for the monitoring procedure was common to each. The core functionality for the protocol that is common to both modalities is shown in Table 1. From the end user's perspective, the two modalities differ only in the way the participant interacts with the device. These differences are summarized in Table 2. The native smartphone application was specifically developed for Android mobile phones (Figures 1 and 2). Android is an operating system created by Google, which runs on mobile phones from different manufacturers. Although this software was developed to run on any Android phone, for this study we used the Orange San Francisco device. For the purpose of this study, it was not wirelessly enabled and all answers were stored on the mobile phone handset for downloading at the end of the sampling procedure. However, this made no difference to the user's perspective of the software. The SMS text-only implementation was driven by openCDMS [14], an open source, secure online platform, which facilitated both the sending of questions and the storing of responses (Figure 3). openCDMS is a Web-enabled server-side application written in Java developed for electronic data collection in clinical studies and trials. It had pre-existing capabilities for SMS text messaging, individual participant records, and study questionnaires. Where new functionality was needed for the text message system, it was specifically developed. BODY.METHODS.SEMISTRUCTURED INTERVIEWS: The PANSS [13] was conducted by an experienced administrator before and after each period of sampling. The PANSS is a semistructured interview assessing positive (7 items), negative (7 items), and global (16 items) symptoms of psychosis and has been extensively validated [13,15]. In this study, the PANSS was employed to provide convergent validity to the adapted mobile phone assessment depression items and to determine which form of delusions should be assessed. BODY.METHODS.QUANTITATIVE FEEDBACK QUESTIONNAIRE: A purpose-designed quantitative feedback questionnaire was developed to assess the acceptability and feasibility of the native smartphone application and SMS text-only implementations (see Tables 3 and 4). This included reactivity to the methodology and whether it had been successfully integrated into an individual's everyday routine. Three items were taken from a previous study assessing the use of PDAs in individuals with psychosis [16]. These were "Overall, this was stressful", "Overall, this was challenging", and "Overall, this was pleasing". All items were rated from 1 (Not at all) to 7 (Very much so). In order to gauge the feasibility of the two methods of mobile phone assessment in the long-term management of patient's symptoms, participants were also asked to the maximum length of time within which they hypothetically would be willing to complete questions with each delivery modality. At the end of the study, participants were also asked which delivery modality they found easier to use and which they preferred (smartphone application, SMS text-only implementation, or no preference). Table 1 Core functionality common to both the native smartphone application and text-message systems. Configurable number and times of question sets each day At the start of the study, this can be configured for the desired number of questions sets and the times of these questions. During this study, 4 question sets per day were used. Configurable questions The wording of the questions is set up at the start of the study. It is easily configurable to support multiple studies with different questions. In addition, delusion questions are configured at the point when the researcher meets with the participant, through an administrative dialogue. Multiple question sets Multiple sets of questions are supported, and the software will switch between these at each consecutive alarm. So, if there are 2 question sets, it will ask set 1 at the first alarm, set 2 at the second, set 1 at the third, etc. The only exception to this is if the participant fails to answer some of the questions. In that case, the same question set is presented again at the next alarm. Question branching The next question displayed to the user can depend on the answer to one or more previous questions. This allows the questions asked to match the participant’s symptoms or situation. For example, if a participant does not endorse the first question about a particular psychotic symptom, all remaining questions about it will be skipped. This means that the participant does not waste time answering unnecessary questions. Questionnaire timeout A time window is enforced, within which the questionnaire has to be completed. No further answers will be accepted outside of this time window. Logging The time at which each question was answered is recorded to the nearest second. This can be used to analyze the time taken to answer each question as well as the time to complete the whole questionnaire. Branching logic A range of different branching logic types is available. In the following list, the first three items apply to branching based on an answer to a single question. All other items are applicable when branching is based on answers to multiple questions. Less than Greater than Equal to Greater than sum Less than sum One is less One is greater All are less All are greater Table 2 Human-machine interface difference between native smartphone application and SMS text-only implementations of the common diagnostic assessment. Native smartphone application SMS text message Alerts Reuses Android’s Alarm Manager so user definable alerts are available; delivered at semirandom intervals during the data collection period; users can snooze the alert to be reminded 5 minutes later. Only a single alert for each question set. Alert is the phone’s SMS alert, triggered when a SMS question is received; delivered at semirandom intervals during the data collection period. As each question is delivered as an SMS text message, an alert is triggered for each question. A reminder SMS is sent after 5 minutes if no response is received. Questions Presented as one question per page in the application. User is able to navigate through the pages of questions. Delivered as SMS text messages one question per SMS. User must respond with an SMS text message and wait for the next question in the set. Data input Continuous slider bar, user slides with finger touching screen. Position of slider mapped to 7-point Likert scale. User enters number between 1 and 7 as a response. Saving data Fully automatic, no user input required User must send SMS text message containing the response value in reply to the question to record their response. Figure 1A typical question from the Android app implementation, showing the full screen with the question and the slider for data entry. Figure 2The start screen shown to the user in the Android app at the start of each set of questions (from which the users may proceed or delay ["snooze"] for a further 10 minutes they wish). Figure 3SMS configuration screen implemented in openCDMS. BODY.METHODS.DIAGNOSTIC ASSESSMENT ITEMS: Seven different symptom dimensions were assessed using previously validated mobile phone assessment scales. In order to minimize the burden on participants, these were split into two alternating sets. In set one, hopelessness (2-4 items), depression (2-6 items), and hallucinations (2-8 items) were assessed; and in set two, anxiety (1-4 items), grandiosity (2-3 items), paranoia (3-6 items), and delusions (0-8 items) were examined. Therefore, although there were four assessment points, each set of questions was assessed only twice per day. All questions related to the period of time that had elapsed since the last entry. Items were branched so that the questions changed depending on an individual's previous responses. The depression subscale was adapted from the original validation study in an effort to increase its association with the PANSS depression scale, since the correlation was moderately low. The new subscale consisted of the items: "I have felt miserable" (new), "I have had no interest in seeing other people" (new), "I have felt worthless" (new), "I have felt sad", "My mood has affected my appetite or sleep", and "I have had thoughts about harming myself". As a result, the correlation between the mobile phone assessment scale and the PANSS was increased from rho = 0.45 (P=.01) in the original research to rho = 0.56 (P=.01) in the current study (calculated for the week of sampling with the native smartphone application). A strong correlation indicates that the interview and mobile phone–based assessments are tapping into similar concepts. The adapted depression assessment scale also showed considerable variability across time (mean squared successive difference: 2.2 (SD 2.9); within participant standard deviation: 0.8 (SD 0.6), suggesting that it was sensitive to subtle fluctuations in mood. A wide range of delusions have been reported in the psychosis literature [17,18]. Therefore, during the initial briefing session, the researcher selected which delusion items were presented to the participant through the administrative dialogue. The choice of items was based on consultation with clinical staff and an initial PANSS interview to assess symptoms. For those individuals experiencing three or greater delusions, those two with the greatest conviction and distress were entered. The frequency of the different delusions were: "I have felt like other people were reading my thoughts" (n=5), "I have felt that my thoughts were being controlled or influenced" (n=4), "I have felt like I could read other people's thoughts" (n=3), "I have felt like things on the TV, in books or magazines had a special meaning for me" (n=3), "I have felt like something bad was about to happen" (n=2), "I have felt distinctly concerned about my physical health" (n=2), and "I have felt like my thoughts were alien to me in some way" (n=1). The delusion items were kept constant for each participant across the two conditions of the trial. BODY.METHODS.PROCEDURE: A randomized repeated measures crossover design was employed. Participants were randomly allocated to either completing 6 days of sampling using the native smartphone application implementation on a smartphone provided to them for the purpose of the study, or via SMS text-only implementation using their own phone. There was then a 7-day rest period in order to reduce carryover effects before the individual completed a further 6 days of sampling with the alternate delivery modality. Each participant, therefore, completed two periods of sampling: 1 week with the native smartphone application implementation and 1 week with the SMS text-only implementation. Randomization was achieved through the openCDMS software. openCDMS uses a permuted block randomizer, and in this case we used a minimum block size of 4 and maximum of 6. The researcher initially met with participants to obtain written consent and demographic information, complete clinical interviews, provide training in the first delivery modality, and administer practice questions. The participant number and delusion items were set by the researcher on either a password-protected administrators' page (on the native smartphone application) or through the openCMDS website (on the SMS text-only implementation). The volume of the alarm prompts was also set according to the preference of the participant when using the native smartphone application implementation. On each day of sampling, participants could complete a maximum of four sets of questions, available at pseudorandom times (selected by a random number generator at least 1 hour apart) when prompted by the mobile phone device between 09:00 and 21:00 hours. All participants had 15 minutes from the first alarm within which to complete the questions. A forced entry time was thought to prevent a self-selection bias (ie, answering the questions only when asymptomatic) [19]. The researcher telephoned once or twice (as per the participant's preference) during the week in order to encourage compliance, answer questions, and to ascertain any problems with the software. The researcher attempted to keep the number of calls made to participants balanced over the two conditions: smartphone: mean, 1.7 (SD 0.5); text-messages: mean, 1.7 (SD 0.6). Upon completion of the first week of sampling, the researcher met with the participant to re-administer the clinical interviews and to gather quantitative feedback on the first device. This procedure was then repeated the following week with the alternate device. At the end of the study, participants rated which device they preferred and found easier to use. Qualitative interviews were also conducted (the results of which are provided in a separate manuscript). Participants on pay-as-you-go tariffs received £50 worth of phone credit, which they topped up prior to the week of text-message questions. If the participant was on a contract tariff (ie, direct debit) but did not have unlimited free texts, then they were reimbursed £50 at the end of the study. All participants were reimbursed an additional £30 upon completion of both sampling procedures. BODY.METHODS.STATISTICS: All analysis was conducted in Stata 10.0 [20]. First, it was important to determine whether there was an interaction between period and delivery modality allocation. Delivery modality order (smartphone application then SMS text-only implementation or SMS text-only implementation then smartphone application) was entered into regression analysis as a predictor of the total score for each of the three outcome measures (mean time taken for each entry, number of completed data points, or quantitative feedback score) summed across the two conditions. A Spearman correlation was used to assess the similarity between scores across the two conditions. Subsequent analysis was performed in a nested (long) form of the data. This data structure violates the assumption of independent observations meaning that additional constraints need to be placed on the statistical models. Multilevel modelling (xtreg) was therefore used to investigate whether delivery modality (smartphone application or SMS text-only implementation) or time-point (week 1 or week 2) significantly predicted any of the outcome variables. The outcomes were the mean time taken to complete each entry, the number of completed data points, and quantitative feedback scores. As the highest level of clustering, participant number was entered as the random effect. Bootstrapping was used to account for the use of non-normal distributed variables in all analysis. This has been suggested as an appropriate alternative when parametric assumptions are not met [21]. The variables were manually standardized in order to aid the interpretation of the results. BODY.RESULTS: We were not able to keep a systematic record of how many individuals were approached by their care coordinators for participation and declined. However, it was the impression of clinical teams that the refusal rate was around 30%. Of the 38 individuals who were referred to the research team, 8 changed their mind, 3 were unable to be contacted, and 3 were deemed ineligible prior to consent. This provided a final sample of 24 individuals, all of whom completed the feedback assessments (ie, no participants withdrew from the study). One individual, however, did ask for the SMS text-only assessment to be ended 2 days early because she found it was making her ruminative. BODY.RESULTS.TESTING FOR AN INTERACTION BETWEEN SAMPLING PERIOD AND METHOD OF ASSESSMENT: First, we assessed whether there was an interaction between the sampling period and device type. Regression analysis showed that the order of the two conditions did not significantly predict the total number of entries an individual completed (β =.06, SE.22, P=0.78), nor the length of time it took to complete each entry (β = -.06, SE.20, P=.78). However, starting with the SMS text-only implementation did show nonsignificantly increased negative appraisals of the devices (β = -.35, SE.20, P=.08). BODY.RESULTS.COMPARING NATIVE SMARTPHONE APPLICATION AND SMS TEXT-ONLY IMPLEMENTATION: The number of entries completed during sampling was strongly correlated between the two delivery modalities (rho = 0.61, P=.002). When controlling for order effect, participants completed significantly greater numbers of entries on the native smartphone application when compared to SMS text-only implementation (Tables 3 and 4). Past studies have defined compliance as completing at least one-third of all possible data -points [22]. Using this criterion, 88% (n=21) of individuals were compliant when using the native smartphone application, whereas 71% (n=19) were compliant when using SMS text-only implementation. Participants completed a significantly greater number of entries in week one (mean = 16.4, SD 16.4) when compared to week two of sampling (mean = 13.7, SD 6.5; β = -.22, SE=.11, P=.04). When broken down by day, participants completed a mean of 3.4 (SD 0.8) entries on day 1, 3.8 (SD 0.5) on day 2, 3.0 (SD 1.2) on day 3, 3.4 (SD 1.1) on day 4, 3.0 (SD 1.3) on day 5, and 3.1 (SD 1.2) entries on day 6 when using the smartphone application. When using the SMS system this was lower at 2.3 (SD 1.4) entries on day 1, 2.4 (SD 1.3) entries on day 2, 2.6 (SD 1.5) entries on day 3, 2.6 (SD 1.3) entries on day 4, 2.0 (SD 1.4) entries on day 5, and 1.9 (SD 1.7) entries on day 6. The length of time taken to complete each entry was highly correlated between the delivery modalities (rho = 0.62, P=.02), suggesting that those individuals who took longer on the native smartphone application also took longer to complete the SMS text-only implementation. However, as can be seen in Tables 3 and 4, individuals took an average 68.4 seconds (SD 39.5) to complete a full set of questions on the native smartphone application and 325.5 seconds (SD 145.6) on the SMS text-only implementation (β = 0.78, SE 0.09, P<.001). Thus, questions sets took 4.8 times longer on the SMS text-only implementation, than on native smartphone application. There was no significant difference in the length of time (seconds) it took participants to complete entries in week 1 (mean = 210.4, SD 171.3), when compared to week 2 (mean = 183.5, SD 166.3; β = -.08, SE=.09, P=.36) of sampling. The total quantitative feedback scores for each device were also strongly correlated (rho = 0.48, P=0.02), suggesting that appraisals of the procedure were similar across both delivery modalities. No significant difference was observed for the total quantitative feedback score when comparing the two delivery modalities. Although none of the individual quantitative feedback items were significantly different, nonsignificantly higher scores (suggesting greater agreement) were observed for SMS text-only implementation on the items "Were there times where you had to stop doing something in order to answer the questions?", "Were there times when you felt like not answering?", and "Was filling in the questions inconvenient?" (Table 3). When considering an order effect, participants scored higher on the items "Were there times when you felt like not answering?" (week 1 mean 2.2, SD 1.1), week two mean 3.1, SD 2.2; β = 0.26, SE=.12, P=.03) and "Was it difficult to keep the device with you or carry it around?" (week 1 mean 1.8, SD 1.2; week 2 mean 2.6, SD 1.9; β = 0.26, SE=.12, P=.03) in the second, when compared to the first week of sampling. No other significant order effects were observed on the quantitative feedback scores. Participants also reported the maximum length of time within which they would hypothetically be willing to complete questions on each delivery modality, which is displayed in Table 5. At the end of the final period of sampling, 67% (n=16) of individuals preferred the native smartphone application, 13% (n=3) of people preferred SMS text-only implementation, and 21% (n=5) of people had no preference. Additionally, 71% (n=17) of individuals found the native smartphone application easier to use, 17% (n=4) of people found SMS text-only implementation easier to use, and 13% (n=3) of people had no preference. Worthy of note is that 2 out of the 3 individuals who preferred their own phone currently owned a smartphone, which they used to complete the SMS text-only implementation. Table 3 Quantitative feedback scores for the native smartphone application and SMS text-only implementation, and summary statistics.   Native smartphone application SMS text-only implementation       Mean SD Min Max Mean SD Min Max β P SE                       Time taken to complete questions (seconds) 68.4 39.5 18.8 179.7 325.5 145.6 118.8 686.9 0.78 <.001 0.09 Number of entries completed 16.5 5.5 4.0 24.0 13.5 6.6 0.0 24.0 -0.25 .02 0.11 Did answering the questions take a lot of work? 1.8 1.1 1 5 2.3 1.6 1 6 0.17 0.13 Were there times when you felt like not answering? 2.3 1.3 1 5 3.0 2.1 1 7 0.21 .07 0.12 Did answering the questions take up a lot of time? 1.7 0.9 1 4 2.3 1.6 1 7 0.24 0.14 Were there times where you had to stop doing something in order to answer the questions? 3.4 1.7 1 7 4.1 1.7 1 7 0.20 .10 0.12 Was it difficult to keep track of what the questions were asking you? 1.6 1.2 1 7 1.9 1.7 1 7 0.11 0.15 Were you familiar with using this type of technology? 4.7 2.3 1 7 5.3 2.2 1 7 0.14 0.14 Was it difficult to keep the device with you or carry it around? 1.9 1.4 1 6 2.4 1.8 1 6 0.16 0.12 Did you ever lose or forget the device? 1.7 0.9 1 4 1.8 1.4 1 6 0.06 0.13 Was using the key pad/touch screen difficult to use? 2.0 1.3 1 5 1.8 1.4 1 6 -0.08 0.15 Do you think other people would find the software easy to use? 5.3 1.8 2 7 5.9 1.4 3 7 0.19 0.16 Do you think you could make use of this approach in your everyday life? 4.0 1.8 1 7 3.9 2.2 1 7 -0.02 0.13 Do you think that this approach could help you or other service users? 5.3 1.9 1 7 5.6 1.2 3 7 0.11 0.15 Overall, this experience was stressful. 1.8 1.1 1 5 1.8 1.3 1 6 -0.04 0.18 Overall, this experience was challenging. 2.2 1.6 1 7 2.7 1.7 1 6 0.16 0.13 Overall, this experience was pleasing. 3.7 2.0 1 7 3.7 1.7 1 7 0.01 0.11 Did filling in the questions make you feel worse? 1.8 1.1 1 5 2.1 1.4 1 5 0.14 0.15 Did filling in the questions make you feel better? 2.8 1.5 1 6 3.0 1.6 1 7 0.08 0.13 Did you find the questions intrusive? 2.2 1.2 1 4 2.6 1.8 1 7 0.15 0.14 Was filling in the questions inconvenient? 2.0 1.0 1 4 2.5 1.4 1 5 0.23 .09 0.14 Did you enjoy filling in the questions? 3.6 2.0 1 7 3.7 1.6 1 7 0.01 0.12 Total quantitative feedback score (positive items reversed): 53.0 11.2 33 76 56.2 14.2 27 88 0.13 0.10 Table 4 Quantitative feedback scores for the native smartphone application and SMS text-only implementation–momentary assessment symptom scores. Momentary assessment symptom scores Native smartphone application SMS text-only implementation Mean SD Min Max Mean SD Min Max Hallucinations 2.7 1.8 1.0 6.5 2.5 1.8 1.0 6.0 Anxiety 2.8 2.0 1.0 6.3 2.1 1.4 1.0 6.4 Grandiosity 2.3 1.5 1.0 6.0 2.3 1.4 1.0 6.0 Delusions 2.0 1.3 1.0 5.1 1.9 1.1 1.0 4.7 Paranoia 2.9 1.8 1.1 6.5 2.6 1.7 1.0 7.0 Hopelessness 3.2 1.4 1.0 5.7 3.0 1.3 1.0 5.1 Table 5 Maximum length of time willing to complete questions on the two implementations. Maximum length of time willing to complete questions Smartphone Text messages n (%) n (%) U<2 weeks 2 (8%) 3 (13%) 2-3 weeks 10 (42%) 10 (42%) 3-4 weeks 1 (4%) 5 (21%) 4-5 weeks 3 (13%) 1 (4%) 5+ weeks 8 (33%) 5 (21%) BODY.DISCUSSION: The objective of this study was to compare two different delivery modalities of the same diagnostic assessment for individuals with nonaffective psychosis—a native smartphone application employing a graphical, touch user interface against an SMS text-only implementation. The overall hypothesis of the study was that participants with serious mental illness would find both systems feasible and acceptable to use, measured by the quantitative postassessment feedback questionnaire scores, the number of data points completed, and the time taken to complete the assessment. We also predicted that participants would complete a significantly greater number of data points, in less time, when using a native smartphone application when compared to the text messages and that the former software would be more positively appraised. The length of time that participants would hypothetically be willing to complete questions was also assessed in order to gauge the feasibility of longer-term assessment. In line with the hypotheses, participants completed a significantly greater number of entries on the native smartphone application (69%, mean = 16.5), when compared to the text message interface (56%, mean = 13.5). It is desirable to keep missing data to a minimum in order to generate a representative picture of an individual's symptoms over time. Other studies have observed similarly high rates of compliance to the native smartphone application when using PDAs (ie, 69-72%) [7,23]. The increased usability and streamlined, graphical interface seen in purpose built software applications may encourage greater levels of compliance than those of SMS text-only implementations. On average, sets of questions on the SMS text-only implementation took 4.8 times longer to complete when compared to the native smartphone application, which may have contributed to the reduced rates of compliance seen with this method of sampling. Although just failing to reach statistical significance, the quantitative feedback highlighted greater disruption to activities and inconvenience and less inclination to complete the questions when participants used the SMS text-only implementation. This is perhaps understandable given the greater time investment incurred by this method. Past research has suggested that the movement of mobile technology from the foreground to the background of an individual's life represents an important step in the accommodation of these technologies [24], which may be less likely if it perceived as burdensome. Somewhat surprisingly, the two delivery modalities did not differ in any of the other quantitative feedback items or the total quantitative feedback score. The mean scores suggest that these appraisals were generally positive across both conditions. Thus, both forms of technology were deemed acceptable and well integrated and may represent suitable methods for facilitating real-time assessment. However, in the forced choice questions, the majority of the sample stated that they preferred the native smartphone application and that they found it easier to use, suggesting that this may be a more attractive delivery modality. Of the three individuals who preferred the SMS text-only implementation, two currently owned a smartphone, which they used for completing the text messages. These mobile phones may have allowed for a more streamlined text system (eg, threaded messages) reducing the differences between the two types of devices. While the mean quantitative feedback scores suggested generally positive appraisals of both delivery modalities, these varied considerably between individuals. For example, some participants stated that they felt this technology could help them, whereas others were more skeptical about its advantages. Some participants reported mild negative reactivity to the method. It is possible that mobile phone assessment is less suitable in certain subgroups of patients, and in its future application, reactivity should be carefully monitored. As this technology makes the transition from research to real-world clinical application, it will be vital to assess the feasibility and uptake of this software over longer periods of time and the factors influencing nonparticipation and withdrawal. In this study, participants completed fewer entries in the second week of sampling. Additionally, only a third of participants said they would be willing to complete the procedure for 5 or more weeks with the native smartphone application, with an even lower percentage for the SMS text-only implementation (21%). In the future, it may be necessary to employ machine learning in order to tailor the choice of questions and sampling rates to the service user [25], while placing particular emphasis on symptoms of primary concern to their clinical team. Person-tailored sampling could increase the feasibility of conducting longer-term real-time assessment. Automated and clinician-delivered feedback, or monetary incentives, may also promote acceptance and compliance. In conclusion, this study provides data to suggest that both native smartphone applications and SMS text-only implementation represent acceptable technologies for facilitating real-time assessment in individuals with nonaffective psychosis. However, the native smartphone application was found to be preferable to SMS text-only implementation in terms of greater data point completion and shorter response times. Limitations of this study include the relatively modest length of the sampling procedure and moderate rates of nonparticipation by those approached to take part. In the future, it will be important to upload software applications onto individual's own phone rather than issuing them with an additional device.

TITLE: Oxaliplatin and Bolus-Modulated 5-Fluorouracil as a Second-Line Treatment for Advanced Pancreatic Cancer: Can Bolus Regimens Replace FOLFOX When Considered for Second Line? ABSTRACT: Objective. Comparing activity of 2 regimens combining oxaliplatin to bolus modulated fluorouracil as second line treatment in advanced pancreatic adenocarcinoma pretreated with gemcitabine-containing schedule. Methods. Forty eight patients with advanced pancreatic adenocarcinoma were randomly assigned to receive either FU 500 mg/m2 IV bolus weekly ×6 weeks plus leucovorin 500 mg/m2 IV weekly for 6 weeks during each 8-week cycle plus oxaliplatin 85 mg/m2 IV on weeks 1, 3, and 5 of each 8-week (FLOX) OR receive weekly intravenous infusions of oxaliplatin 40 mg/m2, 5-FU 500 mg/m2, and leucovorin 250 mg/m2 (3 weeks on, 1 week off). Results. Non progression(PR+SD) was found in 33.5% for first regimen and 29% for second regimen, and 37.5% had clinical benefit (FLOX regimen) compared to 50% in 3-weeks regimen. The median TTP was 3.9,4 months respectively. Median OS was 8, 9 months for both regimens. Only one case in 3-weeks arm suffered from grade IV diarrhea. Two cases > grade 2 neutropenia were observed; one in each treatment groups. Grade 3 anemia was recorded in 3 patients (2 in FLOX arm, one in 3-weeks arm). Conclusions. Both regimens showed encouraging efficacy, acceptable toxicity, and clinical benefit. BODY.1. INTRODUCTION: Due to the fact that the majority of pancreatic cancers are unresectable upon diagnosis, curative intent is rarely a goal of treatment, rather increasing survival, time to progression, and quality of life are more realistic goals. Without treatment, median survival for patients with an advanced stage of disease ranges from 3 to 4 months, whereas in patients receiving chemotherapy with single-agent gemcitabine, median survival times between 4.9 and 7.2 months have been reported in randomized phase III studies [1]. Gemcitabine has been the solo player in the field of pancreatic cancer, treatment after replacing 5-FU since 1997, and is still regarded as one standard of care for the first-line systemic chemotherapeutic treatment of patients with advanced pancreatic cancer worldwide. So far, only two randomized phase III trials have demonstrated a significant prolongation of survival with the use of gemcitabine-based combination therapy with either erlotinib or capecitabine [2]. Eventually, progression will occur and the real challenge will be how to treat a patient with advanced pancreatic cancer failing to respond or progressing after gemcitabine. There is no evidence-based treatment recommendation for these patients. The National Comprehensive Cancer Network guidelines for pancreatic adenocarcinoma currently recommend second-line chemotherapeutic treatment after gemcitabine failure in selected patients using, for example, single-agent capecitabine or a combination therapy of fluorouracil, leucovorin and oxaliplatin (FOLFOX-) like regimen [3]. To date, there is no large randomized trial confirming the survival advantages of second-line chemotherapy over best supportive care, yet the preliminary results from a small randomized German study comparing BSC alone versus 5-FU, folinic acid, and oxaliplatin plus BSC after gemcitabine failure showed a prolongation of median survival by approximately 2.6 months with the use of chemotherapy (2.3 versus 4.9 months) [4]. These data were supported by a Japanese study that reported a median survival time of approximately 1.9 months after failure of first-line gemcitabine in 74 patients with pancreatic cancer (of whom 97% received no second-line treatment) [5]. Many protocols containing oxaliplatin, 5FU, and leucovorin, as FOLFOX, FLOX, and 3-week bolus 5FU plus leucovorin and oxaliplatin, are known. Preclinical data suggested that the 5-FU plus oxaliplatin combination is more cytotoxic when 5-FU is given as a short exposure [6], which gives a rationale for exploring the toxicity and efficacy of such protocols in advanced pancreatic cancer. In the current study, we conducted a randomized trial to compare two protocols; FLOX and the 3-weeek bolus protocol regarding toxicity, response rate, and time to progression as primary end points, then overall survival as secondary endpoints. BODY.2. PATIENTS AND METHODS: Patients with advanced unresectable or metastatic pancreatic adenocarcinoma were enrolled under the following Eligibility criteria. Inclusion Criteria. (I) Patients with histologically or cytologically proven locally advanced or metastatic pancreatic adenocarcinoma, (II) with at least 1 bidimensionally measurable lesion (World Health Organization (WHO) criteria); (III) Eastern Cooperative Oncology Group (ECOG) PS of 1-2; (IV) tumor progression after first line gemcitabine (whether gemcitabine pretreated or gemcitabine resistance); (V) absence of severe uncontrolled cardiovascular, metabolic, infectious, or neurological diseases; (VI) adequate bone marrow reserve (neutrophil count > 1.5 × 109/L, platelet count > 100.000/mm3 and Hb > 10 g/dL); (VII) adequate liver function (serum bilirubin < 1.5 mg/dL, serum transaminases < 2x the upper limit of normal); (VIII) adequate renal function (serum creatinine < 1.5 mg/dL); (IX) and age between 18 and 75 years. All participating patients were required to give written informed consent, and ethical approval from MOC committee was obtained before the start of the whole procedure. Exclusion Criteria Histologic types other than adenocarcinoma.Neuropathy ≥ CTCAE grade 1.Ototoxicity > CTCAE grade 2.Serious, active comorbidity, including any of the following: unstable angina and/or NYHA class II–IV congestive heart failure requiring hospitalization within the past 12 months, transmural myocardial infarction within the past 12 months, acute bacterial or fungal infection requiring IV antibiotics, chronic obstructive pulmonary disease exacerbation or other respiratory illness requiring hospitalization or precluding study therapy, hepatic insufficiency resulting in clinical jaundice and/or coagulation defects, active gastrointestinal (GI) ulcers, GI bleeding, inflammatory bowel disease, or GI obstruction, Inadequately controlled hypertension, defined as systolic BP > 150 mm Hg and/or diastolic BP > 90 mm Hg on antihypertensive medications, serious cardiac arrhythmia on medication (well-controlled atrial fibrillation on medication allowed), and history of hypertensive crisis or hypertensive encephalopathy. BODY.2. PATIENTS AND METHODS.2.1. PRETREATMENT EVALUATION: All patients were subjected to staging procedures consisted of medical history, physical examination, echocardiography, serum chemistry panel, complete blood picture, CEA, and CA 19-9. Extent of disease was determined by chest X-rays, computed tomography and/or nuclear magnetic resonance, and endoscopy as needed. Patients underwent followup examinations until death. BODY.2. PATIENTS AND METHODS.2.2. RANDOMIZATION PROCEDURES: Patients were randomly assigned to one of the treatment regimens (block randomization at 4), where 24 patients were enrolled for each treatment group. BODY.2. PATIENTS AND METHODS.2.3. TREATMENT.2.3.1. FLOX REGIMEN: Oxaliplatin 85 mg/m2 was administered as a 2-hour infusion before LV and FU on days 1, 15, and 29 of the treatment cycle. LV 500 mg/m2 was administered as a 2-hour intravenous infusion weekly for 6 consecutive weeks (on days 1, 8, 15, 22, 29, and 36 of the treatment cycle), followed by a 2-week rest period. FU 500 mg/m2 was administered as an intravenous bolus 1 hour after the LV infusion was begun and was administered weekly for 6 weeks (on days 1, 8, 15, 22, 29, and 36 of the treatment cycle), followed by a 2-week rest period. BODY.2. PATIENTS AND METHODS.2.3. TREATMENT.2.3.2. 3-WEEK BOLUS REGIMEN: 2-hour intravenous infusion of oxaliplatin 40 mg/m2 was followed by bolus leucovorin 250 mg/m2 and bolus 5-FU 500 mg/m2. Each course consisted of weekly administrations for 3 consecutive weeks followed by a week of rest. Therapy continued until disease progression, unacceptable toxicity, patient's refusal, or a maximum of 6 courses. All patients received intravenous dexamethasone 8 mg, Ondansitrone 8 mg as antiemetic prophylaxis. Therapy was withheld in case of a platelet count of less than 100.000/mm3 or a neutrophil count of less than 1.500/mm3 or for bilirubin greater than 1.5 times the upper reference level (URL) or transaminases greater than 3 times the URL. During the entire study period, patients received full supportive care to control pain or other symptoms, with careful recording of the treatment. BODY.2. PATIENTS AND METHODS.2.4. TOXICITY: Adverse events were graded according to the National Cancer Institute common toxicity. Oxaliplatin was reduced in the event of persistent paresthesia/dysesthesia between cycles or with pain lasting for >7 days according to staff physician's decision. When paresthesia/dysesthesia with either pain or functional impairment persisted between cycles, Oxaliplatin was discontinued. BODY.2. PATIENTS AND METHODS.2.5. EVALUATION AND STATISTICAL METHODS: Measurable disease response was assessed by RECIST criteria [7]. Partial response (PR), stable disease (SD), and progressive disease (PD) were determined according to these criteria. The sum of PR and SD was reported as disease control rate (DCR). OS was estimated from the date of first treatment to the date of death or the last followup. Clinical benefit assessment was based on patients and physician-reported improvement of cancer-related symptoms and/or stabilization of improvement of PS. The TTP was calculated from the first treatment infusion to the first objective evidence of disease progression assessed by CT scan measurements or early death or date of clinical deterioration and patient not assessable for response. All patients with at least 1 chemotherapy administration were assessed for toxicity. Efficacy assessments were performed on patients who received at least 1 course of therapy. TTP and OS since the start of treatment were estimated on an intent-to-treat basis and analyzed according to the Kaplan-Meier method. Comparison between survival curves was done through log rank test to estimate P value utilizing GraphPad prism version 5 software. The required number of patients for this phase II study was determined according to a Jehan phase II optimal design [8] for a goal of 20% true clinical benefit; with α- and β-error probability of 0.05 and 0.20, respectively, an accrual of 24 patients assessable for response was planned. BODY.3. RESULTS: Forty-eight patients with unresectable or metastatic pancreatic cancer pretreated with gemcitabine (including gemcitabine resistance or gemcitabine pretreated) in Ain Shams University Hospitals were included along the period between October 2008 and September 2011. The patients' characteristics encountered in the current study were outlined in Table 1. Median age for both groups was 56 years and 54 years, respectively. Sixteen males out of total twenty-four cases were encountered in FLOX arm compared to 17 in 3-week bolus arm. Thirty patients in both groups received prior Gemcitabine as a single agent. BODY.3. RESULTS.3.1. TOXICITIES: Grade 3 or 4 toxicities experienced by at least 5% of patients according to treatment arm are summarized in Table 2. Only one case in 3-week arm suffered from grade IV diarrhea. Two cases of neutropenia exceeding grade 2 (but no febrile neutropenia) were observed; one in each treatment groups. Grade 3 anemia was recorded in 3 patients (2 in FLOX arm, one in 3-week arm). Most nonhematological side effects were less than grade 3. BODY.3. RESULTS.3.2. EFFICACY: No complete response was registered among all assessable 48 patients throughout the study duration for FLOX regimen, three patients (12.5%) had partial response, five patients (21%) had stable disease, and three out of 8 patients with pain at presentation (37.5%) had clinical benefit. The median time to progression was 3.9 months (95% CI, 2–4.6) (range: 1.5–5.5). Median survival time was 8 months (95% CI, 4.0–12). For 3-week regimen, two patients (8%) had partial response, five patients (21%) had stable disease, and four out of 8 patients with pain at presentation (50%) had clinical benefit as shown in Table 3. The median time to progression was 4 months (95% CI, 1.8–5) (range: 1.2–6). Median survival time was 9 months (95% CI, 3.5–13) as shown in Figures 1(a) and 1(b). There was no statistical significance in progression-free survival between the 2 regimens (P value by log rank test = .4619), and so was the situation in overall survival (P-value by log rank test = .5248). BODY.3. RESULTS.3.3. COST COMPARISON: Although it was not planned as a target for the current study, yet it was an interesting issue to compare cost of chemotherapy per patient for every 8 weeks of treatment for each regimen. For FLOX regimen this cost was approximately 1200 USD versus 1400 USD for the 3-weeks regimen (due to mainly the amount of discarded oxaliplatin in every injection time that was more in the second regimen) as in Table 4. BODY.4. DISCUSSION: Advanced pancreatic cancer remains a rapidly lethal cancer, with a median survival of 6 months with currently approved therapies [8]. The role of second-line chemotherapy after failure of first-line therapy in such cases is not well established, but a theoretical possibility exists in which salvage chemotherapy after the failure of first-line treatment may influence the survival. For the scale of patients with good performance status, progressing after first-line gemcitabine therapy, NCCN recommends fluoropyrimidine-based chemotherapy [9]. But for time being there is still a debate to treat or best supportive care? A phase III trial after failure of first-line gemcitabine compared BSC plus with biweekly oxaliplatin combined with weekly 5-FU as 24 hours infusion plus leucovorin, versus BSC alone [4]. After the first 46 patients out of 165 planned, the BSC arm had to be closed because BSC alone was no longer accepted by participating centers, with a possible survival benefit for second-line chemotherapy: 21 weeks (95% CI; .7; 23.3) versus 10 weeks (95% CI; 7.7; 12.3). So a second question is as the following: what is the best option of treatment? Adding oxaliplatin to continuous infusion fluoropyrimidine as a second-line salvage therapy for this category has been investigated in some phase II trials [10–12]. In a series of unselected patients the FOLFOX4 regimen yielded a 14% PR rate with 38% of patients showing SD for a DCR of 57%. Median duration of PR was 5.2 months, while median time to progression and overall survival was 4 and 6.7 months, respectively [10], but all the regimens of continuous infusion necessitate either hospitalization or pump application with their financial load upon health care system. This was the rationale to investigate regimens including oxaliplatin and bolus fluorouracil, with the theoretical premise of being as active as continuous infusion regimens, as well simpler in administration, less in cost, and better in toxicity profile. In the current study, two regimens of oxaliplatin and bolus fluorouracil have been investigated, FLOX regimen that was used in metastatic colorectal cancer with adequate efficacy and acceptable toxicity profile [12]. The current study revealed nonprogression (PR + SD) in 33.5% for first regimen and 29% for second regimen, and 37.5% had clinical benefit (FLOX regimen) compared to 50% in 3-week regimen. The median time to progression was 3.9 months and 4 months, respectively. Median survival time was 8 months and 9 months for both regimens, respectively, with no statistically significant difference in progression-free or overall survival. Regarding toxicity, only one case in 3-week arm suffered from grade IV diarrhea. Two cases of neutropenia exceeding grade 2 (but no febrile neutropenia) were observed; one in each treatment group. Grade 3 anemia was recorded in 3 patients (2 in FLOX arm, one in 3-week arm). Most nonhematological side effects were less than grade 3. So it is the time for the 3rd question; are these results comparable to those of FOLFOX regimens (infusion fluorouracil)? Gebbia et al., 2007, carried out a retrospective study including 42 patients who received standard FOLFOX4 regimen biweekly until progression or unacceptable toxicity. The study revealed six partial responses (14%) and 16 stabilizations (38%) were recorded for a tumor growth control rate of 57%. The median time to progression (TtP) was 4 months (range 1–7 months), and median overall survival (OS) was 6.7 months (range 2–9 months). A stabilization of performance status (PS) and a subjective improvement of cancer-related symptoms was recorded in 27 patients [13]. The good nonprogression rate in this study may be attributed to the high percentage of responding patients in this study to the first line therapy (50%) compared to 12% in our study. Tsavaris et al., 2005, in a prospective Phase II study evaluated a second-line combination regimen of oxaliplatin together with leucovorin-modulated 5-FU in 30 patients and revealed an encouraging response rate of 23% with a corresponding disease-control rate of 53%. Median overall survival was 5.8 months in this patient population [14]. These data correlate well with the retrospective analysis from Italy, which found a response rate of 14% together with a disease-control rate of 52% with the use of a FOLFOX-4 regimen in gemcitabine-pretreated patients [14]. Another phase II trial of oxaliplatin plus capecitabine in a series of 41 patients reported a PR in one case and SD in eight patients with a median OS of 5.8 months, and a 6-month and 1-year survival rate of 48% and 22%, respectively [9]. Toxicity was, however, significant. Preliminary results of another trial of OXP/5-FU in a series of 23 patients have shown an OS of 4 months [15]. Novarino et al., 2009, in a study on 23 gemcitabine pretreated patients with advanced pancreatic cancer revealed no objective response in all 17 assessable patients and 4 patients had stable disease, whereas 13 had tumor progression. Median duration of stable disease was 14 weeks. Median time to progression of disease (TTP) was 11.6 weeks. Seven patients experienced grade 3-to-4 toxicity. The regimen was associated with 36% clinical benefit [16]. In conclusion, combining oxaliplatin to bolus fluorouracil (either in FLOX or 3-week regimens) as a second line in gemcitabine pretreated patients with advanced or metastatic pancreatic adenocarcinoma showed encouraging efficacy, acceptable toxicity, and some clinical benefit specially when palliation or good quality of life is a target keeping in mind the simplicity in administration, the no need for hospitalization, and the less financial load specially with FLOX. Further studies with large number of patients investigating the efficacy and tolerability of such bolus regimens in gemcitabine-pretreated pancreatic cancer patients are warranted.

TITLE: A controlled study of community-based exercise training in patients with moderate COPD ABSTRACT.BACKGROUND: The effectiveness of clinic-based pulmonary rehabilitation in advanced COPD is well established, but few data exist for less severe patients treated in alternative settings. The purpose of this study was to investigate whether a novel, community-based exercise program (CBE) was feasible and effective for patients with moderate COPD. ABSTRACT.METHODS: Nineteen patients with moderate COPD (mean FEV1 62%) and self-reported exercise impairment were randomized to 12-weeks of progressive endurance and strength training at a local health club under the guidance of a certified personal trainer, or to continuation of unsupervised habitual physical activity. Outcomes assessed at baseline and 12 weeks included session compliance, intensity adherence, treadmill endurance time, muscle strength, dyspnea, and health status. ABSTRACT.RESULTS: Compliance was 94% and adherence was 83%. Comparisons between CBE and control groups yielded the following mean (SEM) differences in favor of CBE: endurance time 134 (74) seconds versus -59 (49) seconds (P = 0.041) and TDI 5.1 (0.8) versus -0.2 (0.5) (P < 0.001). The CBE group increased muscle strength (weight lifted) by 11.8 kilograms per subject per week of training (P < 0.001). SGRQ was not significantly changed. ABSTRACT.CONCLUSIONS: We demonstrated the feasibility and effectiveness of a novel community-based exercise program involving health clubs and personal trainers for patients with moderate COPD. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT01985529. BODY.BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a progressive debilitating disease that leads to worsening symptoms and declining exercise capacity [1]. Progressive weight loss is also common [2] and associated with increased mortality and compromised quality of life [3]. Skeletal muscle dysfunction is recognized to account for an appreciable portion of the exercise impairment in COPD patients [4]. Evidence for this particular abnormality includes reduced muscle aerobic enzyme content [5,6] and a tendency to develop premature lactic acidosis during exercise [1]. Furthermore, calf muscle cross-sectional area is reduced compared with controls [7] and muscle strength is also impaired [8]. Importantly, COPD is now regarded as preventable and treatable [9]. One could argue that successful treatment requires earlier recognition of the decline in exercise performance and implementation of a structured exercise program to reverse its deleterious effects. Structured exercise training is strongly endorsed for COPD in the context of a pulmonary rehabilitation program recognizing that these programs increase functional capacity, decrease symptoms, reduce utilization of health-care resources and improve quality of life [10-12]. The Global Initiative on Obstructive Lung Disease (GOLD) recommends that patients with COPD should be referred for pulmonary rehabilitation once their FEV1 falls below normal [13]. This equates to the transition from mild to moderate disease and corresponds with the time when patients begin to significantly reduce activities of daily living [14]. Furthermore, the American Thoracic Society/European Respiratory Society Statement on Pulmonary Rehabilitation suggests all patients suffering from respiratory disease associated with diminished functional capacity and reduced health related quality of life may benefit from pulmonary rehabilitation [12]. While pulmonary rehabilitation is practiced as a multi-disciplinary therapy, evidence-based analysis identifies exercise training as the most effective component [15]. Despite guidelines, a national survey of 283 pulmonary rehabilitation programs in 1995 identified considerable differences in program content [16]. Lack of consistency in the approach to aerobic and resistance training in chronic pulmonary disease is likely to result in sub-optimal outcomes in many cases. A paradigm shift is needed to make exercise training programs for COPD patients more scientifically rigorous and cost-effective. We have studied the feasibility of a community-based exercise program under the supervision of personal trainers appropriately certified in exercise physiology and trained to manage pulmonary patients safely and with confidence. We examined the hypothesis that in patients with moderate COPD and reduced aerobic capacity, the combination of aerobic exercise training plus nutritional counseling would result in improvement in exercise endurance, muscle strength, activity levels and quality of life compared with control group having only nutritional counseling. A secondary aim was to demonstrate the feasibility of community-based exercise rehabilitation delivered through health clubs and personal trainers at an earlier stage in the progression of COPD. BODY.METHODS.SUBJECTS: We recruited 19 men and women with moderate COPD as defined by the GOLD [17] criteria (FEV1/FVC, % <70%; FEV1 < 70% and >50% predicted), a 10 pack-year smoking history and self-reported functional impairment. Exclusion criteria included current smokers, pulmonary diseases other than COPD, use of supplemental oxygen, musculoskeletal disease that impaired exercise performance and unstable coronary artery disease or congestive heart failure. The study was approved by the UCLA Office for the Protection of Research Subjects (IRB#11-000175) and all subjects gave written informed consent. BODY.METHODS.STUDY DESIGN: This was a small parallel-group, randomized controlled study to assess the feasibility of implementing a community-based exercise program for patients with moderate COPD (see Figure 1). At the screening visit, spirometry was performed to assess eligibility in terms of the GOLD staging followed by body plethysmography, diffusing capacity and a maximal, symptom-limited, incremental exercise test on a treadmill ergometer using a standardized protocol [18]. Then, after a resting period of 2 hours, they performed a constant work-rate exercise test at 90% of the maximum work rate calculated from the incremental test. Figure 1Study design showing time frame and procedures at each visit. Within two weeks of the screening visit, subjects returned to repeat the constant work-rate exercise test also on a treadmill at the same constant work rate as at the first visit. This was intended to overcome any learning effect, and the best value for endurance time from the two constant load tests was used for future comparison. Subjects had assessments of body composition, dyspnea (Baseline Dyspnea Index, BDI) and quality of life (St. George's Respiratory Questionnaire, SGRQ) [19,20]. Although not felt to be malnourished, all subjects were given nutritional guidance from a certified nutritionist by way of best clinical practice and also so as to be able to offer some benefit to those subjects randomized to the control group. The nutritional counseling was based on a Food Frequency Questionnaire [21] administered every two months to assess diet and specifically to ascertain the habitual protein intake of the subject. Detailed, written recommendations were then given on an individualized basis with the goal of achieving a daily protein intake of 1.5 g per kilogram of body weight plus a daily energy intake of 35 kilocalories per kilogram of body weight. Subjects were then randomized 1:1 into two groups: (a) community based exercise program (CBE) or (b) usual habitual activity (control). Both groups continued standard COPD management based on published guidelines and appropriate for the stage of their disease. Subjects randomized to exercise training had measures of lower extremity strength by 1-repetition maximum (1-RM) for leg press at the time of the first training session. At 12 weeks, all patients were asked to repeat baseline measurements. Minor exacerbations, defined as an increase in two or more symptoms (dyspnea, sputum volume and sputum purulence) for three or more days [22] but not requiring hospitalization, were referred to the subjects' primary care physician for intensification of bronchodilator therapy and/or an antibiotic. Details of minor exacerbations were recorded but subjects continued in the study. Major exacerbations, requiring use of systemic corticosteroids or hospital admission, resulted in withdrawal from the study. BODY.METHODS.COMMUNITY-BASED EXERCISE PROGRAM: Following randomization, those subjects assigned to the exercise program were assigned to a personal trainer operating out of one of three local health clubs. They met at mutually convenient times, twice per week for 12 weeks. Each of the clubs provided facilities for aerobic exercise and resistance training. Two facilities provided two trainers each and a fifth trainer was in solo practice. All five trainers possessed Bachelors and/or Masters degrees in exercise science or related fields. Three were -Certified Strength and Conditioning Specialist (CSCS) from the National Strength and Conditioning Association and two held Exercise Specialist certifications from the American College of Sports Medicine. Specific methodologies were thoroughly reviewed with each personal trainer prior to the study along with general information regarding COPD and its effects on exercise tolerance and physical function. Initial, individualized aerobic and exercise prescriptions were developed for each subject based on findings from the maximal incremental exercise test performed at the screening visit. Information derived from this test was also used to define a safe ceiling for exercise intensity for each individual subject. Each session included a warm-up and cool-down period. Subjects then performed 30 minutes of continuous aerobic exercise in the target heart rate range based upon an initial exercise prescription developed by the investigators. The trainers were entrusted with progressing the exercise prescription at reinforcement visits in order to maintain the appropriate exercise intensity. Subjects also performed resistance training designed upon basic principles, starting with one set of each of eight conventional free-weight or machine-based exercises for the large muscle groups of the upper and lower body and progressing to two sets (typically after 10-12 sessions) at the discretion of the personal trainer. The stipulated goal was to maintain 12 repetitions of each exercise in correct form before muscle fatigue (typically 70-75% of the maximal 1-RM weight). Subjects assigned to the control group were told to continue their activities of daily living. They were contacted by telephone every month to see assess their progress. BODY.METHODS.OUTCOMES: The co-primary outcome measures were endurance time for the constant work rate exercise test and change in muscle strength as calculated by total weight lifted per week. Secondary outcomes were lean body mass, percentage body fat, dyspnea (Transition Dyspnea Index, TDI) and SGRQ. Compliance was calculated as the percentage of sessions attended (out of 24), and adherence was calculated as the percentage of time subjects spent exercising in or above their target heart rate zone. BODY.METHODS.STATISTICAL ANALYSIS: The sample size for this study was estimated using an anticipated effect size for change in exercise endurance time derived from experience with bronchodilator therapy and other interventions in COPD patients. For example, acute administration of inhaled ipratropium bromide increased endurance time by 1.1-2.8 minutes and more recent work with the long-acting bronchodilator tiotropium bromide increased exercise endurance time for a similar work rate protocol by 105 seconds compared with placebo [23]. Importantly, changes in endurance time of this magnitude were felt to be associated with clinically meaningful perceptions of improvement on the part of the investigators and the patients. In a study of repeated constant work rate exercise testing in severe COPD patients (FEV1 = 40%) in clinically stable state, O'Donnell et al [24] showed that exercise endurance time was highly reproducible with a standard deviation of 60 seconds. From our database of submaximal CWR tests in moderate COPD patients repeated during the same week, we found a standard deviation for endurance time of 88 seconds. Our power analysis, based on a standard deviation of 88 seconds, α = 0.05 and 10 subjects in each group indicates a power of 86% (β = 0.14) to detect a 120 second difference in endurance time between the two groups after 3 months. We used a two-sample t-test to evaluate whether exercise endurance time was longer in the subjects treated with exercise training and nutritional counseling versus nutritional counseling alone. We also carried out regression analysis to adjust for baseline covariates including age, gender and habitual activity level. Similar statistical methods as employed for the primary outcome measure were also applied to compare body mass index, TDI and SGRQ between groups. BODY.RESULTS: Between August 2008 and December 2010, 29 patients were consented for the trial. Five patients failed screening. Twenty-four patients were randomized. Five patients in the exercise group withdrew from the study after randomization (one with worsening knee pain, one with an unrelated ankle injury, one with exacerbation of COPD, one with newly diagnosed lung cancer and one who was lost to follow-up). The baseline characteristics for both the control and exercise groups are listed in Table 1. The two groups were balanced in terms of age, anthropometrics, body composition, resting pulmonary function and breathlessness. The average FEV1 of all subjects was 62%, indicating moderate severity of COPD. Those randomized to the exercise group completed 202 of 216 total sessions, resulting in 94% compliance with the CBE. These subjects performed endurance exercise in or above the prescribed target heart rate zone 83% of the time. Table 1 Baseline characteristics of the control and community-based exercise groups   Control group CBE group No. of subjects 10 9 Age (year) 72.0 (10.1) 66.8 (8.1) Male sex (%) 60 33 Height (m) 1.70 (0.1) 1.72 (0.1) Weight (kg) 76.2 (10.9) 76.6 (23.4) FEV 1 (%) 60.8 (10.9) 63.6 (7.6) Resting heart rate (/min) 82.7 (8.3) 76.4 (9.9) O 2 saturation (%) 96 (2) 96 (2) BDI 6.5 (0.6) 6.6 (0.9) Body fat (%) 27.1 (8.2) 32.5 (3.1) Lean body mass (kg) 55.8 (10.4) 56.4 (16.7) Values are mean (standard deviation). FEV 1 : forced expiratory volume in one second. O 2 saturation: oxygen saturation. BDI: Baseline Dyspnea Index. m: meters. s: seconds. kg: kilograms. As shown in Table 2 and Figure 2, at the end of 12 weeks, endurance time increased 134 seconds in the CBE group and decreased 59 seconds in the control group (P = 0.041). Patients who participated in the CBE arm lifted significantly more weight at the end of the exercise program compared with the beginning as shown in Figure 3: 3432 versus 1775 kilograms (P < 0.001). This represents a 93% overall improvement over 12 weeks, averaging an increase of 11.8 kilograms of weight lifted per subject for every week of exercise training. Figure 4 shows that SGRQ total scores decreased by 0.7 in the control group and 4.6 in the CBE group (P = 0.611). Whilst the differences between groups were not significant, the average fall in SGRQ total score within the exercise training group exceeds the MCID of 4 units which was not the case with the control group. TDI focal score, shown in Figure 5, increased by 5.1 in the CBE group versus a decrease of 0.2 in the control group, indicating less breathlessness (P < 0.001). There was no difference in body composition as determined by skin folds between the CBE and control group after intervention (change in body fat -1.1% versus -0.1%, P = 0.285; change in lean body mass +1.2 kilograms versus +0.1 kilograms, P = 0.206). Table 2 Change in exercise performance, quality of life and dyspnea in control patients versus those in the community-based exercise program   Control group CBE group   P-value   Pre Post Change Pre Post Change   Endurance time (s) 408 (67) 350 (89) -59 444 (121) 578 (147) +134 0.041 SGRQ total score 35.1 (3.8) 34.4 (6.2) -0.7 32.1 (5.4) 27.6 (4.6) -4.6 0.611 TDI focal score NA -0.2 (0.5) -0.2 NA 5.1 (0.8) +5.1 <0.001 Total weight lifted (kg/week) NA NA NA 1775 (303) 3432 (361) +1657 <0.001 Values are mean (SEM). SGRQ: St. George’s Respiratory Questionnaire. TDI: Transitional Dyspnea Index. s: seconds. Kg/wk: kilograms per week. NA: not applicable. Figure 2Mean treadmill endurance times for the two groups at baseline and after 12 weeks. Mean changes were a decrease of 59 seconds for the control group compared with an increase of 134 seconds for those completing the community-based exercise program (P = 0.041). Figure 3Changes in total weight lifted for individual subjects from baseline to completion of training. Total weight lifted per week is calculated as the product of load (kilograms) × repetitions × sets for two days in week one and two days at end of study. Diagonal lines represent individual changes. Horizontal lines represent group means and error bars are SEM (P < 0.001). Figure 4Mean SGRQ scores for the two groups at baseline and after 12 weeks. Mean changes were a decrease of 0.7 for the control group and a decrease of 4.6 for those completing the community-based exercise program (P = 0.611). Decreased scores represent improvement in quality of life where a change of 4.0 is considered clinically meaningful. Figure 5Mean TDI focal score for the two groups after 12 weeks. Mean changes were a decrease of 0.2 for the control group and an increase of 5.1 for those completing the community-based exercise program (P < 0.001). Increased scores represent improvement in dyspnea where a change of 1.0 is considered clinically meaningful. BODY.DISCUSSION: We successfully conducted this small, randomized, feasibility study of a community-based exercise program versus habitual activity in patients with moderate COPD. We found increases in exercise endurance time and muscle strength, along with improved health-related quality of life, and reduced dyspnea in those who underwent exercise training compared to those who received nutritional counseling only. The average improvement in endurance time was 193 seconds at 90% of maximum work rate at baseline which compares favorably with changes in endurance time between 100 and 120 seconds in therapeutic trials of inhaled bronchodilators [23,25-27]. Furthermore, compliance and adherence to this community-based exercise program surpassed published rates for clinic- or hospital-based exercise programs [28]. We were unable to demonstrate a statistically significant and clinically meaningful difference in health status between the two groups as measured by SGRQ total score. There are likely two reasons for this: (1) with relatively small numbers of subjects the study was underpowered to prove the SGRQ changes statistically significant and (2) the difference between the groups was narrowed by a Hawthorne effect in the control group. Taking into account these shortcomings of our analysis, we feel that the reduction of SGRQ of -4.6 units in the training group is likely a real effect and brings our results, in terms of the effects of an exercise program on health status, into better alignment with the results of other studies. Other studies on community based exercise programs have demonstrated improvements in functional capacity, dyspnea, and disease specific quality of life [29-32]. However, these studies have focused on patients with severe COPD (FEV1 < 50%) whereas our subjects were less severe (mean FEV1 62%). We specifically sought to follow GOLD [33] and ATS/ERS [12] guidelines by recruiting patients with less severe disease. We reasoned that these patients would find a community-based exercise program, close to their home or workplace, more convenient than the typical clinic-based or hospital-based program. We also surmised that less severe patients would feel comfortable exercising in a health club environment alongside apparently normal individuals. The compliance with recommended training sessions of 99% attests to the success of this strategy. Furthermore, knowing that supervised exercise training is significantly more successful than unsupervised training, we utilized personal trainers to improve subject adherence to exercise prescriptions. The 83% adherence to the target heart rate range for aerobic training is impressive and clearly associated with marked improvements in the prespecified outcome measures of exercise performance. We are aware of two other studies that examined outpatient rehabilitation in patients with moderate COPD. Cambach et al [34] found improved exercise tolerance and quality of life in patients with moderate COPD after 6 months of rehabilitation, however, this sample of patients also included asthmatics whose disease state might have been more fluctuant. Additionally, this rehabilitation program was conducted in various medical practices under the guidance of clinically trained physiotherapists, rather than in a local health club with certified personal trainers as in our community-based program. In another study, van Wetering, et al[35] randomized 199 patients with moderate COPD (mean FEV1 60%) to an interdisciplinary community-based COPD management program (INTERCOM) or usual care. This study was conducted in the Netherlands where community-based physical therapists can be assigned to visit patients in their homes. The investigators reported improvements in health related quality of life, breathlessness, exercise performance, muscle strength, and body composition, but were unable to show improvements in muscle strength. Although this rehabilitation program was conducted in the community, it utilized clinically trained physical therapists and other medical personnel. Despite the promising data in favor of community based rehabilitation programs, it remains clear that there is not yet any consensus regarding the ideal structure for such programs. The traditional medical model of pulmonary rehabilitation is focussed on exercise encouragement in more severely disabled patients. The goals are often limited to tolerance of dyspnea and modest gains in exercise performance are considered acceptable. Our program is the first to use certified personal trainers, based in health clubs, which are becoming increasingly prevalent in the community. This health club model is more directed to maximizing performance using basic principles of reconditioning and muscle strengthening. Whilst there is undoubtedly considerable overlap in these approaches, we think that the personal trainers can potentially bring a new dimension to pulmonary rehabilitation. The major limitation of our study was its small size. However, this was a pilot study and one of the main purposes was to demonstrate the feasibility of CBE training in moderate COPD. Our results should prompt further research to standardize procedures and define optimal interventions. A further limitation is that, although subjects were randomly assigned to two groups (exercise training or usual physical activities), the individual subjects and the trainers obviously could not be blinded to their treatment allocation. We attempted to reduce observer bias by having the baseline and final assessments performed in the research laboratory, distant from the training location, with the technician blinded and specifically instructed not to inquire about group allocation. Our exercise program was novel, but there was incomplete standardization of the exercise facilities involved in the study and the variability in the training and experience of the personal coaches. The exercise facilities included a nationally distributed chain, a small privately-owned facility, and a large privately-owned facility. The exercise coaches were all certified personal trainers and they received an educational session and information on exercise limitations in COPD patients. A future study might attempt to standardize the approach to training even further. BODY.CONCLUSIONS: In summary, our data confirm that community based exercise programs are not only feasible, but also effective, in patients with moderate of COPD. Such programs improve functional capacity, dyspnea, and health related quality of life, and should therefore be considered in the routine management of these patients. Furthermore, our findings contribute to a growing body of evidence that calls for a paradigm shift in the philosophy of pulmonary rehabilitation. As a treatment strategy, community-based exercise programs should be introduced earlier in the progression of COPD so as to forestall the development of comorbidities associated with the lack of regular exercise and deconditioning. Future efforts should be made to establish uniform guidelines to ensure that community-based exercise training programs for COPD patients are scientifically rigorous and cost-effective. BODY.ABBREVIATIONS: COPD: Chronic obstructive pulmonary disease; GOLD: Global initiative on obstructive lung disease; FEV1: Forced expiratory volume in one second; FVC: Forced vital capacity; BDI: Baseline dyspnea index; SGRQ: St. George's Respiratory Questionnaire; CBE: Community-based exercise; 1-RM: 1-repetition maximum; TDI: Transition dyspnea index; ATS/ERS: American Thoracic Society/European Respiratory Society; INTERCOM: Interdisciplinary Community-based COPD Management Program; SEM: Standard error of the mean. BODY.COMPETING INTERESTS: The community-based exercise training program was implemented at Equinox Fitness Club, Century City, CA. TWS and CBC serve as an uncompensated members of the Equinox Health Advisory Board. None of the other authors have any financial or non-financial competing interests. BODY.AUTHORS’ CONTRIBUTIONS: SA drafted the manuscript. MA was involved in subject testing, data collection and analysis. MQ was involved in data collection. TWS was involved in the study design, assisted with data collection, and participated in manuscript preparation. C-HT performed the statistical analysis. CBC conceived the study, was involved in its design, assisted with data analysis and participated in manuscript preparation. All authors read and approved the final manuscript. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2466/14/125/prepub

TITLE: ABSTRACT: Recent evidence suggests that the fat mass and obesity-associated gene (FTO) genotype may interact with dietary intakes in relation to adiposity. We tested the effect of FTO variant on weight loss in response to 2-year diet interventions. FTO rs1558902 was genotyped in 742 obese adults who were randomly assigned to one of four diets differing in the proportions of fat, protein, and carbohydrate. Body composition and fat distribution were measured by dual-energy x-ray absorptiometry and computed tomography. We found significant modification effects for intervention varying in dietary protein on 2-year changes in fat-free mass, whole body total percentage of fat mass, total adipose tissue mass, visceral adipose tissue mass, and superficial adipose tissue mass (for all interactions, P < 0.05). Carriers of the risk allele had a greater reduction in weight, body composition, and fat distribution in response to a high-protein diet, whereas an opposite genetic effect was observed on changes in fat distribution in response to a low-protein diet. Likewise, significant interaction patterns also were observed at 6 months. Our data suggest that a high-protein diet may be beneficial for weight loss and improvement of body composition and fat distribution in individuals with the risk allele of the FTO variant rs1558902. BODY: The prevalence of overweight and obesity has increased substantially in the U.S. and worldwide, and the health burden of obesity-related complications has grown accordingly (1–3). Obesity is primarily determined by both genetic and lifestyle factors, including diet, as well as their interactions (4). In the past few years, genome-wide association studies (GWASs) have identified a group of genetic loci associated with BMI and obesity risk (5–7). Among them, the fat mass and obesity-associated gene (FTO) locus shows the strongest effect (5,8). Accumulating evidence has suggested that this locus is involved in the hypothalamic regulation of appetite and dietary energy intake (9,10). Recently, several studies have examined the effect of the FTO-diet interaction on body weight, but the results are not entirely consistent. Several cross-sectional studies showed that dietary factors such as low fat intake might modify the genetic effect of FTO on BMI or fat distribution (11–13). However, the gene by diet interaction was not univocally observed in randomized intervention trials (13–17), although one study found that a Mediterranean diet intervention modified the association between the FTO variant and weight changes in a population with high cardiovascular risk (18). These intervention trials, however, largely are limited by relatively small sample size or the short term of follow-up. In addition, animal studies have suggested that FTO might differentially affect various body compositions and fat distribution at different depots (19–21). Few studies have evaluated systematically the effect of the FTO variant on these measurements. The POUNDS LOST Trial thus far is the largest 2-year randomized intervention trial that tested the effect of four diets varying in proportions of fat, protein, and carbohydrate on weight loss in overweight or obese subjects (22). By use of the data from this trial, we evaluated whether various weight-loss diets might modify the effect of the FTO variant on weight loss and long-term changes in body composition and fat distribution. BODY.RESEARCH DESIGN AND METHODS.STUDY POPULATION.: The POUNDS LOST Trial was conducted from October 2004 through December 2007 at two sites as follows: Harvard School of Public Health and Brigham & Women's Hospital in Boston, Massachusetts; and the Pennington Biomedical Research Center of Louisiana State University System, Baton Rouge, Louisiana. The design and sample collection have been described previously in detail (22). In brief, the study population was composed of 811 overweight or obese (BMI ranged from 25 to 40 kg/m2) participants aged 30–70 years. Major criteria for exclusion were the presence of diabetes or unstable cardiovascular disease, the use of medications that affect body weight, and insufficient motivation as assessed by interview and questionnaire. Among the 742 participants who were genotyped successfully, 61% were women, 80% were white, 15% were black, 3% were Hispanic, and 2% were Asian or other ethnic groups by self-report. The participants were assigned randomly to one of four diets constituting a two-by-two factorial design; the target percentages of energy derived from fat, protein, and carbohydrate in the four diets were 20, 15, and 65%; 20, 25, and 55%; 40, 15, and 45%; and 40, 25, and 35%. After 2 years, 645 participants (80% of total population) completed the trial. The study was approved by the human subjects committee at each institution and by a data and safety monitoring board appointed by the National Heart, Lung, and Blood Institute. All participants provided written informed consent. BODY.RESEARCH DESIGN AND METHODS.MEASUREMENTS.: In the morning before breakfast, body weight and waist circumference (WC) were measured on 2 days at baseline; 6, 12, and 18 months; and 2 years. BMI was calculated as weight by height squared (kg/m2). A dual-energy X-ray absorptiometry (DEXA) scan was performed on 50% of a random sample from the total study participants (n = 424), including 242 (57.1%) women, using a Hologic QDR 4500A (Waltham, MA) (23). Total fat mass (kg), total fat-free mass (FFM; kg), whole body total percentage of fat mass (FM%) and percentage of trunk fat were obtained once at baseline, 6 months, and 2 years. Computed tomography (CT) was used in 50% of a random sample from those participants who had DEXA scans, resulting in a sample of 25% of the total participants (n = 195), including 113 (58.2%) women. Total adipose tissue (TAT) mass, visceral adipose tissue (VAT) mass, deep subcutaneous adipose tissue (DSAT) mass, and superficial adipose tissue (SAT) mass within the abdomen were measured by standard methods (24), once at baseline, 6 months, and 2 years. Because of radiation exposure, premenopausal women would not subject themselves to CT scans. A series of eight single-slice images were obtained every 10 cm from 2 below and 5 above the fourth and fifth lumbar vertebrae interspaces. These contiguous cross-sectional images were analyzed, and then the total volume was calculated from the individual slices. In this analysis, we only included data at baseline, 6 months, and 2 years with all the outcomes because the DEXA and CT scans were only performed at these three time points. BODY.RESEARCH DESIGN AND METHODS.GENOTYPING.: DNA was extracted from the buffy coat fraction of centrifuged blood using the QIAmp Blood Kit (Qiagen, Chatsworth, CA). Single nucleotide polymorphism (SNP) rs1558902 was selected because it had emerged as the top variant of FTO locus for BMI and WC in recent obesity-related GWAS (25,26). The SNP was genotyped successfully in 742 of 811 total participants and 603 of 645 participants who completed the trial using the OpenArray SNP Genotyping System (BioTrove, Woburn, MA). Of the 424 participants who received DEXA scans, 391 were genotyped at baseline, and 224 participants who completed the trial were genotyped. Of the 195 participants who received CT scans, 175 were genotyped at baseline and 105 participants who completed the trial were genotyped. The genotype success rate was 99% in available DNA samples. Replicated quality control samples (10%) were included in every genotyping plate with greater than 99% concordance (27). The allele frequency in two major ethnic groups (white and black) was compatible with Hardy-Weinberg equilibrium (P > 0.05). BODY.RESEARCH DESIGN AND METHODS.STATISTICAL ANALYSIS.: The primary outcomes were changes in body weight and WC. Secondary outcomes were changes in body composition including total fat mass, FFM, FM% and percentage of trunk fat, and fat distribution (TAT, VAT, SAT, and DSAT). Data were pooled from the diets for the two factorial comparisons: low protein versus high protein and low fat versus high fat (22). Because the majority of the study population were white (80%), we also analyzed the main effects and interactions among white participants separately. The Hardy-Weinberg equilibrium and comparison of categorical variables at baseline were assessed with χ2 test. Differences in continuous variables at baseline were tested using ANCOVA, with adjustment for age, sex, and ethnicity. The main effects of genotype and diet intervention on outcome changes at 6 months and 2 years were analyzed using general linear regression models, with adjustment for covariates including age, sex, ethnicity, carbohydrate, the baseline value for the respective outcome, and baseline BMI. We excluded individuals with missing measures at each time point in the analysis. Moreover, to analyze the potential interactions between genotype and diet intervention, an interaction product term of genotype-diet was included in the models. In a secondary analysis, we used linear mixed models, with time as a repeated measurement factor, to test genetic associations with the trajectory of changes in outcomes in the participants who provided measurements at baseline, 6 months, and 2 years in each of four diet groups over the 2-year intervention by including genotype-time interaction terms. Because an additive genetic effect was reported in the original large-scale GWAS in which the SNP was identified (25,26), additive models were analyzed for genotype. All reported P values were two-sided and a P value of 0.05 was considered statistically significant. All data were analyzed with SAS version 9.1 (SAS Institute, Inc., Cary, NC). BODY.RESULTS.CHARACTERISTICS OF STUDY POPULATION.: Baseline characteristics of participants according to the FTO rs1558902 genotype are presented in Table 1. The minor allele frequency (MAF; A allele) was 0.402 in the total population. The genotype frequencies were significantly different among the sexes and ethnicities. After adjustment for age, sex, and ethnicity, all variables such as weight, BMI, WC, body composition, and fat distribution had no association with genotype at baseline. Baseline characteristics were similar among participants in the four diet groups (Supplementary Table 1). Likewise, no associations between the FTO genotypes and these measures were observed in the white participants (data not shown). TABLE 1 Baseline characteristics of the study participants according to FTO rs1558902 genotype BODY.RESULTS.EFFECTS OF : After adjustment for age, sex, ethnicity, baseline BMI, and diet groups, no main effects of the FTO rs1558902 genotype on changes in weight or WC were found in any participants at 6 months and 2 years (data not shown). We next examined the genetic effects on changes in weight and WC following a two-factorial design: low versus high fat and low versus high protein. We found that the risk allele (A) was significantly associated with a 1.51-kg greater weight loss in the high-protein group (P = 0.010), but not in the low-protein group, by the end of intervention (2 years). The changes in weight and WC were less significant at 6 months (Table 2). In subgroups treated by different proportions of dietary fat, we did not find significant genetic effects on changes in weight and WC (all P > 0.05; Supplementary Table 2).Similarly, in the white participants, we found the risk allele was associated with a 1.38-kg greater weight loss in the high-protein group at 2 years (P = 0.028), but not in other subgroups (data not shown). TABLE 2 The effects of the FTO rs1558902 genotype on weight, body composition, and fat distribution response to dietary protein intervention BODY.RESULTS.THE : Consistent with the observations of change in body weight, we found that the rs15589002 risk allele (A) was associated with greater loss of total fat, FFM, FM%, and percentage of trunk fat at 2 years in the high-protein group, but not in the low-protein group (Table 2). Tests for genotype-diet protein interaction were significant on changes in FFM and FM% (for interactions, P = 0.034 and 0.049, respectively) adjusted for age, sex, ethnicity, carbohydrate, baseline BMI, and the baseline value of body composition (Fig. 1). At 6 months, we only observed gene by protein diet interaction on changes in FFM (P = 0.008 for interaction; Fig. 1). The risk allele carriers in the high-protein group had greater loss of FFM than noncarriers, but those in the low-protein group had less loss of FFM compared with noncarriers (Table 2). FIG. 1.Interaction between the FTO rs1558902 genotype and dietary protein intervention on changes in total fat (A), FFM (B), FM% (C), and percentage of trunk fat (D) at 6 months and 2 years. P values are adjusted for age, sex, ethnicity, carbohydrate, baseline values for respective outcomes, and baseline BMI. Data included 52 and 60 (TT), 66 and 73 (TA), and 31 and 28 (AA) participants in the low-protein group and the high-protein group at 6 months, respectively (total n = 310), and 34 and 44 (TT), 46 and 61 (TA), and 19 and 20 (AA) participants in the low-protein group and the high-protein group at 2 years, respectively (total n = 224). We did not find significant genetic effect and interactions between the FTO variant and dietary fat intake on changes in body composition in total participants (Supplementary Table 2 and Supplementary Fig. 1). The results in the white participants were similar (data not shown). We further analyzed body fat distribution measured by CT. At 2 years, we found significant interactions between the FTO rs1558902 genotype and protein diet intervention on changes in TAT, VAT, and SAT (for interactions, P = 0.001, 0.012, and 0.002, respectively; Fig. 2). The risk allele (A) was associated with greater loss of TAT and VAT in the high-protein group but with less loss of TAT and SAT in the low-protein group (Table 2). At 6 months, gene-protein interactions were observed on changes in TAT and SAT (for interactions, P = 0.026 and 0.050, respectively; Fig. 2), and the risk allele carriers in the high-protein group had greater loss of TAT and VAT than noncarriers, but those in the low-protein group had less loss of SAT than noncarriers (Table 2). FIG. 2.Interaction between the FTO rs1558902 genotype and dietary protein intervention on changes in TAT (A), VAT (B), DSAT (C), and SAT (D) at 6 months and 2 years. P values are adjusted for age, sex, ethnicity, carbohydrate, baseline values for respective outcomes, and baseline BMI. Data included 17, 18, 18, and 17 (TT); 30, 35, 35, and 30 (TA); and 10, 13, 13, and 10 (AA) participants in the low-protein group and 18, 22, 22, and 18 (TT); 26, 35, 35, and 26 (TA); and 12, 14, 14, and 12 (AA) in the high-protein group for TAT, VAT, DSAT, and SAT at 6 months (total n = 137); and 12, 15, 15, and 12 (TT); 18, 25, 25, and 18 (TA); and 8, 9, 9, and 8 (AA) participants in the low-protein group and 15, 17, 17, and 15 (TT); 23, 30, 30, and 23 (TA); and 8, 9, 9, and 8 (AA) in the high-protein group for TAT, VAT, DSAT, and SAT at 2 years (total n = 105). We did not find significant genetic effects and interactions on changes in fat distribution in subgroups treated by different dietary fat components (all P > 0.05; Supplementary Table 2 and Supplementary Fig. 2).Similar results were found in the white participants (data not shown). BODY.RESULTS.THE TRAJECTORY OF CHANGES IN BODY COMPOSITION AND FAT DISTRIBUTION BY : In a secondary analysis, we used linear mixed models to assess the genotype by time effect over the 2-year trial in those treated by the four dietary compositions. We observed significant genotype-time interactions on changes in total fat, FM%, and percentage of trunk fat in response to the high-protein diet. When assigned to the high-protein diet, participants who carried the AA genotype had greater loss in total fat, FM%, and percentage of trunk fat than those without this genotype. No genotype-time interaction on changes in body composition was found in the low-protein group (Fig. 3). FIG. 3.Changes in total fat (A), FFM (B), FM% (C), and percentage of trunk fat (D) in the low-protein and the high-protein diet groups according to the FTO rs1558902 genotype from baseline to 6 months and 2 years. P values are adjusted for age, sex, ethnicity, carbohydrate, baseline values for respective outcomes, and baseline BMI. Data included 198, 149, and 99 in the low-protein group and 193, 161, and 125 in the high-protein group for body composition at baseline, 6 months and 2 years, respectively. (A high-quality color representation of this figure is available in the online issue.) We also observed significant genotype-time interactions on changes in fat distribution in response to the low- and high-protein diets. Participants with the AA genotype had greater decrease in fat distribution in response to the high-protein diet compared with those without this genotype. In contrast, participants with the AA genotype were associated with less loss in TAT and SAT in response to the low-protein diet (Fig. 4). FIG. 4.Changes in TAT (A), VAT (B), DSAT (C), and SAT (D) in the low-protein and high-protein diet groups according to the FTO rs1558902 genotype from baseline to 6 months and 2 years. P values are adjusted for age, sex, ethnicity, carbohydrate, baseline values for respective outcomes, and baseline BMI. Data included values at baseline and at 6 months and 2 years for 80, 57, and 38 participants, respectively, for TAT and SAT and 89, 66, and 49 participants, respectively, for VAT and DSAT in the low-protein group; and 71, 56, and 46 participants, respectively, for TAT and SAT and 86, 71, and 56 participants, respectively, for VAT and DSAT in the high-protein group. (A high-quality color representation of this figure is available in the online issue.) We found genotype-time interactions on changes in total fat, FM%, and percentage of trunk fat in response to the low-fat diet (Supplementary Fig. 3), but no genotype-time interactions on changes in fat distribution were found in response to the low- or high-fat diets (Supplementary Fig. 4).A similar trend was observed in the white population (data not shown). BODY.DISCUSSION: In the POUNDS LOST Trial, a 2-year, randomized weight-loss intervention, we found that dietary protein intake significantly modified the effect of an FTO variant on changes in body composition and fat distribution. Carriers of the risk allele (A allele) of the rs1558902 genotype had a greater loss of weight and regional fat in response to a high-protein diet compared with noncarriers, whereas an opposite genetic effect was observed regarding changes in fat distribution in response to a low-protein diet. Our data indicate that the modification effects of dietary treatment were more evident with prolonged intervention. We did not observe significant modification of dietary fat intake on the genotype effects. The rs1558902 genotype was reported to show the strongest association with obesity in the European (25,26) and other ethnic populations (28), and it has strong linkage disequilibrium with other obesity-associated FTO variants such as the rs9939609 genotype. In this study, the MAF of the polymorphism in all participants was similar to those in the HapMap CEU population (0.45). At baseline, no significant difference was observed in anthropometrics and metabolic estimates, body composition, or fat distribution across genotypes. The lack of association with baseline BMI is probably largely due to the fact that the participants were all overweight or obese, so that the groups had relatively smaller variances in BMI than the general population. Several cross-sectional studies showed that diets might modify the effect of the FTO variant on obesity, but the data from randomized diet intervention trials are conflicting and limited by small sample size or short term of follow-up (Supplementary Table 3). Two lifestyle intervention studies with follow-up periods of 9 and 12 months did not find significant influence of FTO polymorphisms (rs8050136 and rs9939609) on changes in body weight or fat distribution related to diet among 200 overweight and obese individuals treated by diets with reduced fat and increased fiber or reduced fat and sugar (13,14). In another 10-week, hypo-energetic diet intervention with either low fat or high fat content, the FTO variant had an effect on only changes in resting energy expenditure, insulin release, and sensitivity, not on weight loss (15). Similarly, in the Finnish Diabetes Prevention Study, the FTO variant did not modify weight change by individualized diet intervention with reduced fat and increased fiber during the 4-year follow-up of 255 individuals with impaired glucose tolerance (16). In our study, when macronutrient components of diets were not considered, we found no main effects of the FTO variant on changes in weight and body composition during the intervention. Grau et al. (15,29) reported that dietary fat/carbohydrate content interacted with some genetic variants including the FTO variant on weight reduction or change in obesity-related phenotypes. In our study, we also found significant gene-diet interactions on changes in body composition and fat distribution. However, our data indicate that it is the dietary protein component, rather than dietary fat, that might drive the observed interactions. In previous studies, high-protein intervention has been found to result in a greater weight loss and abdominal fat mass (30–32). Our results suggest that individuals with a certain genetic background may benefit more in weight loss by following a high-protein diet. The mechanism of how protein intake interacts with FTO genotype is unclear. Our data indicate that the genetic effects on certain fat compositions or depots may be more evident than the effects on overall adiposity. Functional studies have shown that the loss or overexpression of FTO in mice led to different changes in fat distribution at different dissected sites (19–21). FTO mRNA expression was fat depot–specific and was found to differ significantly in subcutaneous fat and in visceral fat (33–35). Epidemiological studies also have shown that FTO variants are significantly associated with distribution of fat depots (13,36,37). Taken together, these data suggest that genetic effects of FTO on the change of fat mass at various sites may be different, and changes in anthropometrics may not adequately reflect the effects of an FTO variant. The genetic effect in our study seemed to be more evident at 2 years than that at 6 months. The results were in line with a recent study by Razquin et al. (18) in which it was found that FTO risk allele carriers had the highest weight reduction after 3 years of intervention with a Mediterranean diet compared with several short-term diet interventions (less than 1 year) in which no influence of an FTO variant on weight loss or change in fat distribution was found (13–15,17). Of note, between 6 months and 2 years of intervention in our trial, the participants regained weight. Therefore, it seems that the genetic variant might affect both the reduction and regain of the adiposity measures. These data suggest that the modification effects of diet treatment on an FTO genotype effect are more likely to be identified in long-term interventions. Several limitations need to be considered when interpreting our findings. Even though our study is thus far the largest and longest diet intervention weight-loss trial, the relatively small sample size of the subgroups may limit the power to detect very moderate genetic effects or interactions. We did not adjust for multiple testing according to the recommendation by Rothman (38) and Lai et al. (39) because outcomes and the repeated measurements at 6 months and 2 years were highly correlated in our study. Overadjustment for multiple comparisons may increase the type II error and reduce power to detect significant differences. In addition, the majority of the total participants were white, and further studies are needed to determine whether our findings are generalizable to other ethnic groups. Even though the randomized clinical trial is thought to be the best model to test gene-environment interactions, we acknowledge that replication in diverse populations is needed to verify our findings. In summary, we found that dietary protein intake might modify the FTO variant's effect on changes in body composition and fat distribution. A high-protein diet may be beneficial for weight loss in individuals with the risk allele of an FTO variant. Further studies are warranted to verify our findings and explore the potential mechanisms.

TITLE: Rotary instruments in the treatment of chronic periodontitis: A randomized clinical trial ABSTRACT.BACKGROUND:: The study aimed at comparing the effectiveness of rotary instruments with hand instruments in non-surgical treatment of chronic periodontitis. ABSTRACT.MATERIALS AND METHODS:: Thirty-eight patients with chronic periodontitis, enrolled to this randomized, controlled clinical trial, were divided into two groups. Patients in the control group received scaling and root planing with hand instruments, whereas patients in the test group received treatment with rotary instruments. Clinical assessment by plaque index, bleeding on probing, probing depth, gingival recession, and clinical attachment level was made prior to and at 6 months after treatment. Differences in the clinical parameters were analyzed using the Wilcoxon signed rank test and Mann–Whitney U test. ABSTRACT.RESULTS:: Both treatments resulted in a significant improvement in all clinical recordings, but no differences in any of the investigated parameters were observed at baseline between the two groups. ABSTRACT.CONCLUSION:: Non-surgical periodontal therapy with the tested rotary instruments may lead to clinical improvements comparable to those obtained with conventional hand instruments. BODY.INTRODUCTION: The ultimate objective of all root treatment procedures is to effectively remove plaque and calculus without causing root surface damage and to render the treated root surface biologically compatible with host periodontal tissue. However, complete removal of subgingival calculus with hand or power-driven instruments is difficult to achieve, even when surgical approach is used. Various power-driven and hand instruments are currently available for scaling and root planing. Although hand scalers are frequently used, considerable time and manual dexterity are required for their effective operation.[1] Moreover, hand scalers are unable to reach the deeper root surfaces where the periodontal pockets are more than 4 mm deep.[23] Consequently, ultrasonic scalers have become more widely used in recent years. Although they are simple to use, it is often difficult to achieve smooth and calculus-free root surfaces.[456] Dental plaque adheres more readily to roughened root surface created by the use of ultrasonic scalers.[7] To overcome these challenges associated with the use of ultrasonic scalers and hand instruments, carbide rotary instruments for scaling and root planing have been developed. Some studies suggested that hand instruments produce smoother root surface,[891011] while other studies were in favor of use of ultrasonic instruments to achieve a smooth surfaces, as these were found to be less damaging to the root surfaces than hand curettes.[1213] Clinical studies have shown that attachment gain and pocket probing depth reduction are identical after the use of hand instruments and sonic or ultrasonic scaling.[141516] Most of the data available in literature compare the efficacy of hand curettes and ultrasonic instruments. However, until now, hardly any study is available concerning the clinical outcome following non-surgical periodontal treatment with rotary carbide instruments. Therefore, the aim of the present study is to assess the clinical effectiveness of carbide bur when compared to scaling and root planing with hand instruments. BODY.MATERIALS AND METHODS: All the subjects in the present study were recruited from the Department of Periodontology and Implantology. The ethical committee of Himachal Institute of Dental Sciences approved the study protocol, and all participating patients signed an informed consent at the beginning of the study. BODY.MATERIALS AND METHODS.SELECTION OF SUBJECTS: Thirty-eight patients (20 females and 18 males) with advanced chronic periodontitis (Armitage 1999)[17] were recruited for the study. The inclusive criteria for patient selection were as follows: Adults between 18 and 70 years of ageGood level of oral hygiene [as a criterion for good level of oral hygiene, a mean plaque index (PlI) score <1 was chosen] (Lӧe 1967).[18] The exclusion criteria were: Patients seeking treatment of periodontitis for the last 2 yearsPatients using antibiotics for 12 months prior to treatmentPatients with any systemic diseasePatients using medicated mouth washesPregnancy. All subjects were enrolled in a hygiene program 6 weeks prior to the treatment. They received professional supragingival tooth cleaning with ultrasonic scalers (supragingival ultrasonic tip) and oral hygiene instructions. A supragingival scaling and reinforcement of oral hygiene was performed at baseline as well as 4, 8, 12, 16, 20, and 24 weeks after treatment. BODY.MATERIALS AND METHODS.STUDY DESIGN AND TREATMENT: The present study was conducted between June 2012 and January 2013, and was performed according to a parallel group design. All patients were treated according to a "one-stage procedure" with either (1) the rotary instruments, Desmoclean (Hager, Germany) [Figure 1] or (2) Gracey curettes (Hu-Friedy Co., Chicago, IL, USA) [Figure 2]. Figure 1The rotary instrument along with micro-motor contra-angle handpiece Figure 2Gracey curettes In the test group or rotary instrument group, there were 19 patients (12 females and 7 males). In the test group, carbide burs with non-cutting, elliptical, and hexagonal head were used with micro-motor contra-angle handpiece at 8000 rpm (rotation per minute) with light pressure and water spraying [Figure 1]. In the control group or Gracey curette group, there were 19 patients (8 females and 11 males). Instrumentation for both groups was performed until the root surface was adequately debrided and planed. All the instrumentation was done under local anesthesia by one experienced operator. The curettes were sharpened at the operator's request and no restrictions in instrumentation time were set in any group. BODY.MATERIALS AND METHODS.CLINICAL MEASUREMENTS: The following parameters were recorded at baseline (after 6 weeks pre-treatment phase) and 6 months after therapy: Full mouth plaque score (FMPS) (O'Leary et al. 1972)[19]Probing depth (PD)Gingival recession (GR)Clinical attachment level (CAL)Bleeding on probing (BOP). Presence or absence of bleeding on probing was assessed up to 30 s after probing. The measurements were made at six aspects per tooth – mesio-buccal (mb), mid-buccal (b), disto-buccal (db), mesio-lingual (ml), mid-lingual (l), and disto-lingual (dl) – using a manual periodontal probe. BODY.MATERIALS AND METHODS.RANDOMIZATION OF THE STUDY: Sixty-eight patients underwent a screening examination which included full mouth probing, plaque index, and a radiographic examination. Thirty-eight patients with generalized advanced chronic periodontitis, who fulfilled all the prerequisites, were finally recruited for the present study. This screening test and final recruitment was conducted by examiner (A). These 38 patients were randomly assigned to two treatment groups using random tables. The randomization list was kept by the second examiner (B), and the patients were given sealed envelopes indicating the treatment modality. Now, examiner (C) opened those envelopes and performed the therapy. Examiner (A), who was unaware of the treatment received, performed the recording of all clinical parameters. And finally, examiner (B) compiled the data received from examiner (A) according to the randomization list. At all time points, the outcome of research was assessed blindly. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: The Wilcoxon signed ranks test was used to compare the baseline data to those at 6 months for each treatment group. Comparisons between treatment groups at baseline and those at 6 months were accomplished with the Mann–Whitney U test. The α-error was set at 0.05. The power of study, given 1 mm as the significant difference between the groups, was calculated to be 0.99. BODY.RESULTS.FMPS AND BOP: The FMPS and BOP for both the treatment groups at baseline and 6 months are summarized in Table 1. No statistically significant differences were found between these groups. Table 1 FMPS and BOP: Mean values (in percentage) at baseline and 6 months BODY.RESULTS.CLINICAL PARAMETERS IN SINGLE-ROOTED TEETH: In the rotary instrument group, 312 single-rooted teeth and in the Gracey curette group, 395 single-rooted teeth were treated. At moderately deep pocket (4-5 mm) sites, in the rotary instrument group, pocket depth at baseline (4.5 ± 0.5) reduced to 3.7 ± 1.2, whereas in the Gracey curette group, the pocket depth reduced from 4.5 ± 0.3 to 3.4 ± 1.1 [Table 2]. At deep pocket site (initial PD > 6 mm), in the rotary instrument group, PD reduced from 7.2 ± 1.5 to 6.5 ± 1.8 and in the Gracey curette group, it reduced from 6.7 ± 0.9 to 5.4 ± 1.8. But while comparing these two groups, the results were statistically nonsignificant. Similarly, other clinical parameters like GR and CAL gave statistically nonsignificant results [Table 3]. Table 2 Single-rooted teeth: Mean PD, GR, and CAL (±SD) at baseline and 6 months at sites with initial pocket depths of 4-5 mm ( n =38) Table 3 Single-rooted teeth: Mean PD, GR, and CAL (±SD) at baseline and 6 months at sites with initial pocket depths >6 mm ( n =38) BODY.RESULTS.PARAMETERS IN MULTI-ROOTED TEETH: In the rotary instrument group, 256 multi-rooted teeth and in the Gracey curette group, 302 multi-rooted teeth were treated. Clinical improvement was noticed in both moderately deep pocket sites and deep pocket areas. But comparison of groups showed them to be statistically nonsignificant [Tables 4 and 5]. Table 4 Multi-rooted teeth: Mean PD, GR, and CAL (±SD) at baseline and 6 months at sites with initial pocket depths of 4-5 mm ( n =38) Table 5 Multi-rooted teeth: Mean PD, GR, and CAL (±SD) at baseline and 6 months at sites with initial pocket depths >6 mm ( n =38) BODY.DISCUSSION: In order to provide a root surface compatible for soft tissue healing, the old concept of extensive cementum removal has been questioned by various experimental studies.[2021] Nowadays, the trend in therapeutic methodology in periodontology is minimum aggressive approach in both surgical and non-surgical treatments. In the literature, there are large numbers of studies comparing the effect of hand curettes and ultrasonic scalers on root surface roughness.[456111314] Some studies found that ultrasonic instruments produce rough and irregular root surface as compared to hand curettes,[45] whereas other studies suggested that clean and smooth root surface can be achieved with the use of ultrasonic scalers.[1314] Kishida and Sato stated that ultrasonic instrument produces smooth and regular surface as compared to hand curette.[12] But the use of a diamond bur at high speed increased the risk of root surface damage;[22] similar results were obtained by Evian and Horowitz.[23] Solis Moreno et al. compared the root surface roughness after using Gracey curettes, termination diamond burs (40 μm), a piezoceramic ultrasonic scaler, and a piezosurgery ultrasonic scaler, using confocal microscopy and scanning electron microscopy.[24] They found that the termination diamond burs (40 μm) produced a rougher surface than the ultrasonic instruments and the hand curettes. Whereas Dahiya et al. found a smoother root surface with rotary bur as compared to hand curettes and ultrasonic scalers.[25] The difference in the results of these two studies can be explained on the basis of different characteristics of bur. In the previous study, diamond burs were used, whereas in latter study, non-cutting carbide burs were used. Khatiblou and Ghodossi studied the effect of root roughness on periodontal healing and found that periodontal healing following flap surgery occurred regardless of whether the subgingival root surface was rough or smooth.[26] The present randomized clinical trial evaluated the clinical effectiveness of rotary instrument and hand curettes. In the rotary instrument group, four different carbide burs (I = 23 mm, II = 29 mm, III = 26 mm, and IV = 29 mm) with non-cutting, elliptical, and hexagonal head in four different shaft designs were used according to manufacturer's instructions [Figure 1]. Carbide burs were used at 8000 rpm with light pressure and water spraying, so as to avoid excessive damage due to frictional and thermal insults. Similar parameters were adopted by Dahiya et al. in their study.[25] No restriction in the instrumentation time was set in any group. Instrumentation in both groups was performed until a smooth and hard surface was achieved. Limiting the time might result in under- or over-instrumentation of root surfaces. The smoothness of the root surface was assessed using a periodontal probe (Hu-Friedy PCP 11; Hu-Friedy Co.) and explorer (Hu-Friedy Wilkins-Tufts 17/23; Hu-Friedy Co.). This method of evaluating the root surface smoothness was similar to the method used by Ioannou et al.[16] All clinical parameters were assessed at baseline and after 6 months of therapy because healing following non-surgical therapy is almost complete at 3 months. However, a slower ongoing, but limited healing can continue for a longer time (up to 9 or more months) following the treatment.[27] Ioannou et al.[16] and Sculean et al.[28] also evaluated the clinical parameters at baseline and 6 months after therapy. The results of our study suggested that both hand curettes and rotary instruments are effective treatment modalities for periodontal disease. When interpreting the results, it has been noted that sites where probing depths were initially deep showed more GR, more gain of CAL, and deeper residual PD at 6 months examination than the sites with initial moderate PD. Some clinical studies have demonstrated that the reduction of PD and the improvement of CAL after both surgical and non-surgical periodontal treatments are dependent on the initial PD.[2930] But while comparing carbide bur with hand curettes, in terms of periodontal healing by using PD, CAL, and GR clinical parameters, statistically nonsignificant results were found. The possible explanation for such results could be the enrollment of all the patients in supragingival professional tooth cleaning and oral hygiene reinforcement program at baseline as well as 4, 8, 12, 16, 20, and 24 weeks after treatment. Some clinical studies have shown that root surface debridement with either hand curettes or ultrasonic devices leads to similar clinical and microbial improvement of periodontal conditions.[3132] Oberholzer et al. did not find any significant differences in pocket depth reduction and clinical attachment gain after creating rough or smooth surface during flap operation. This indicates that subgingival roughness does not interfere with healing if there is good supragingival plaque control and that long-term success of this treatment is dependent on the quality of maintenance therapy. But a smooth surface may be advantageous near the gingival margin, since a smooth surface is less likely to accumulate plaque than a rough surface.[33] Because of lack of published data regarding the effect of carbide bur on clinical parameters like PD, CAL, and GR, we could not compare the results of the present study with those of other studies. BODY.CONCLUSION: The present study shows that non-surgical periodontal therapy with both hand curettes and rotary burs was well tolerated as all the treated pockets showed uneventful healing. Moreover, the use of rotary instruments in the treatment of chronic periodontal disease gave comparable results with traditional approach of scaling and root planing carried out with hand curettes. Our study also emphasizes the importance of scaling and root planing for maintenance of good periodontal health. Consequently, it is also suggested that more studies evaluating healing outcome and patients' compliance after both treatment modalities are needed in order to draw definitive conclusion about the stability of each therapeutic approach.

TITLE: Feto-maternal outcomes and Glycemic control in Metformin versus insulin treated Gestational Diabetics ABSTRACT.OBJECTIVE:: To evaluate and compare feto-maternal outcomes and glycemic control in metformin versus insulin treated gestational diabetics. ABSTRACT.METHODS:: The study was conducted in 2010- 2012 as a part of M. Phil at Civil hospital, Lyari General Hospital and Mamji Hospital in Karachi. After written informed consent, 71 GDM diagnosed females with WHO criteria were enrolled. They were divided into two groups. Group-A, 32 females were given oral metformin 500 mg TDS while Group-B, 39 females were given insulin 0.8-0.9 mg/kg/day in two divided doses subcutaneously. Patients were followed till term. Feto-maternal outcomes were evaluated in 25 patients in each group who completed the study. ABSTRACT.RESULTS:: When groups were compared, newborns in Group-B were significantly more in weight (p=0.01). Significant numbers of babies were delivered after 38 weeks of pregnancy in Group-B (P=0.021). There were two intrauterine deaths and significantly higher HbA1C at term in Group-B. (P=0.03). FBS at term was non-significant (p=0.079) and there was more number of cesarean sections due to feto-maternal disproportion in Group-B (28% vs.2%). Results analyzed for glycemic control before and after the treatment revealed that FBS was statistically less in Group-A (p=0.00) whereas for Group-B the value of FBS and HbA1C was statistically high. (p=0.002 & 0.04 respectively). ABSTRACT.CONCLUSION:: Metformin has produced better effects on feto-maternal outcomes and glycemic control in comparison to Insulin in GDM. BODY.INTRODUCTION: Glucose intolerance occurring during the prenatal period, Gestational Diabetes Mellitus (GDM), is usually identified in the second trimester of pregnancy. The causes of pancreatic beta - cell dysfunction in pregnancy, leading to insulin deficiency in GDM, are not fully understood. Main factors are adiposity in pregnancy, autoimmune beta cell dysfunction, decreased insulin secretion and insulin resistance.1 Pathophysiology is strongly suggestive of circulating placental and maternal hormones leading to a decrease in glucose sensitivity by the tissue receptors and subsequent increases in the carbohydrate tolerance. From 1995 to 2005, gestational diabetes had amplified by 45% overall, with the world wide prevalence from 3.0 to 4.4%.2 Women of South Asia are seems to be at the utmost risk for GDM with the prevalence rate being 3.2-3.5%. GDM is almost 72%more frequently seen in patient's age ≥ 30 years, 50% more common with positive family history and 29% occur more in those with raised body mass index.3 Traditionally Insulin has been known as an effective therapy for GDM as it shows adequate control of raised blood glucose levels.4 It was initially prepared as a lifesaving drug for Type-I insulin deficient diabetic patients but later was also used for gestational diabetes as most of the oral medications were considered to be teratogenic. For decades it has been used for the treatment of diabetes in pregnancy.5 It requires proper storage and is associated with high incidence of maternal morbidity and mortality such as maternal hypoglycemia, macrosomic babies leading to assisted or surgical deliveries and more weight gain in pregnancy.6 Metformin, an oral anti diabetic drug is being used since 1960 in patients with Type-2 diabetes mellitus. It has now been upgraded to a Category B drug as it is not associated with any fetal congenital anomalies.7 It was initially used to reduce insulin resistance in females with the polycystic ovarian syndrome and as it showed remarkable results with no side effects in pregnancy so was considered a better alternative for the management of GDM.8 It can be a logical treatment for pregnant females as it produces the euglycemic state by improving insulin sensitivity, reducing hepatic gluconeogenesis and increasing peripheral glucose uptake and utilization.9 Present study was designed to evaluate and compare the effects of metformin and insulin on feto-maternal outcomes and glycemic control in GDM patients. BODY.METHODS: This Karachi based study was started in Lyari General Hospital, Mamji Hospital and Civil Hospital, after obtaining approval from Institutional Review Board and Ethical Committee of Dow University, Karachi. It was conducted during 2010-2012 as a part of M.Phil. For this study, high risk patients were recruited from hospitals after obtaining a written informed consent. High risk patients include those with bad obstetric history (previous abortions, intra uterine deaths or still births), strong family history, obese patients and patients with history of gestational diabetes in previous pregnancies. Seventy one diabetic pregnant females were enrolled in the study in second trimester after confirmation for GDM with Oral Glucose Challenge Test and Oral Glucose Tolerance Test, according to WHO criteria. These were divided randomly into two groups. The odd numbers patients (GROUP A=32 patients) were given Tab. Metformin, 500mg thrice daily, with dose escalation therapy prescribed in once daily dose in morning and then increased to thrice daily dose as required, depending upon the glycemic control of the patients. Even number (GROUP B=39 patients) received subcutaneous insulin. A total Insulin dose was calculated using 0.8 units /kg /day in 2nd trimester and 0.9 units /kg/day in 3rd trimester. 2/3 of the dose was prescribed in the morning and 1/3 of the dose was advised in the night. They were counseled well for strict diet control with provided diet charts (1800-2000 kcal /day) and were also advised for 30 minutes of walk at least thrice weekly. These patients were followed fortnightly up till 32 weeks and weekly till term. Maternal outcomes were then re-evaluated at 36 weeks of pregnancy with fetal outcomes and mode of delivery, being noted soon after the child birth.10 Data was subjected to statistical analysis and results were tabulated using SPSS-16. Chi square, independent T-test and Fisher exact tests were applied accordingly and p-value of 0.05 or more was considered significant. BODY.RESULTS: Results were compiled for 50 patients who completed the study with 25 patients in each group. By completion of study 21 patients had dropped out due to various reasons or delivered elsewhere and were excluded from the study. When maternal characteristics were evaluated no difference was observed between age and weight of the patients in Group-A versus Group-B (p=0.09 & p>0.99 respectively). There were statistical differences present between the fasting blood sugar (FBS) and random blood sugar level (RBS) between the groups (p=0.039 & 0.001 respectively). When glycated hemoglobin (HbA1C) was compared statistically, results were non-significant (P=0.182) (Table-I). Table-I Baseline Maternal Characteristics Group-A v/s Group-B (n=50). S. No. Characteristics Group-A (n=25) Group-B (n=25) p-value Mean ± S.D Mean ± S.D 1. Age (years) 29.76±3.41 31.60±4.27 0.099 2. Weight(kg) 77.90±7.65 77.9 ±9.03 NA 3. FBS-1(mg/dl) 104.40±13.12 117.9 ±29.06 0.039 * 4. RBS(mg/dl) 171.16±37.44 239.16±69.78 <0.001 * 5. HbA 1 C--1 (%) 5.28±0.42 5.43±0.34 0.182 Group-A: Metformin with diet control and exercise, Group-B: Insulin with diet control and exercise * Statistically significant (Independent T-test applied), FBS1: fasting blood sugar level at enrollment HbA 1 C-1: Glycated hemoglobin at enrollment. When fetal outcomes were evaluated the babies of Group-B were significantly more in weight as compared to Group-A (p=0.01). Apgar score was statistically non-significant between the groups. On comparing delivery age, the results were statistically significant and more numbers of babies were delivered after 38 weeks of pregnancy in Group-B (P=0.021). All the babies were healthy and alive in Group-A whereas there were two intrauterine deaths at term in Group-B. When maternal outcomes were tabulated there were significant differences between Group-A and B for HbA1C at term, significantly higher in Group-B (P=0.03). FBS at term was non-significant between the groups (p=0.079). There were more number of cesarean sections in Group-B due to feto-maternal disproportional ratio as babies were bigger and heavier (28% vs.2%) (Table-II). Table-II Fetal and Maternal outcomes Group A v/s B (n=50). Fetal outcomes Group-A (n=25) Group-B (n=25) P- value Mean±SD Mean±SD Weight of the baby (kg) 3.14±0.32 3.44±0.46 0.01 * Apgar score 8.64±0.56 8.22±2.43 0.39 n (%) n (%) Delivery age 0.021 *   37 weeks 19(76%) 11(44%)   38 weeks 6(24%) 14(56%) Condition of baby 0.49@   Alive baby 25(100%) 23(96%)   Intrauterine death 0 2(8%)   Still birth 0 0 Maternal outcomes Mean±SD Mean±SD HbA 1 C-2 (%) (36weeks) 5.42±0.34 5.72±0.35 0.003 * FBS -2 (36 weeks) 93.48±11.9 102.08±20.63 0.079 n (%) n (%) Mode of delivery NA   Normal vaginal 7(28%) 5(20%)   Assisted delivery 2(8%) 3(12%)   Cesarean section 16(64%) 17(68%)   Cesarean due to bad obstetric history 14(56%) 10(40%)  Cesarean due to Feto–maternal disproportion 2(8%) 7(28%) Group-A: Metformin with diet control and exercise, Group-B: Insulin with diet control and exercise, HbA 1 C-2: Glycated hemoglobin at 36 weeks, FBS-2: fasting blood sugar at 36 weeks, * statistically significant result (chi square and independent T test applied), NA: chi-square test not applicable, @: Fisher exact test applied. When results were analyzed for glycemic control before and after the treatment in the same groups it was observed that FBS were statistically less in Metformin treated females (p=0.00). HbAIC values were statistically non-significant before and after the treatment in Group-A. For Group-B the value of FBS was statistically high at term (p=0.002). Even HbA1C levels were statistically more at term in Group-B females (p= 0.04) (Table-III). Table-III HbA 1 C and FBS at enrollment and at 36-37 weeks of pregnancy Group A v/s B (n=50). Groups At enrollment At term P- Value Mean ± SD Mean ± SD Group-A FBS 104.40±13.12 93.43±11.93 0.00 * Group-A Hba 1 c 5.284±0.42 5.420±0.346 0.061 Group-B FBS 117.92±29.06 102.08±20.64 0.002 * Group-B Hba 1 c 5.43±0.345 5.68±0.506 0.04 * Group-A: Metformin with diet control and exercise, Group-B: Insulin with diet control and exercise HbA 1 C: Glycated hemoglobin; FBS: fasting blood sugar, * statistically significant result (Paired T-test applied). BODY.DISCUSSION: Gestational diabetes mellitus is a growing health issue in many parts of the world. Our population is at higher risk to develop this disorder because of genetic, social, and environmental factors.11 Gestational diabetes has serious, long-term consequences for both baby and mother. Early diagnosis and management can surely improve the outcomes for these women and infants.11 Base line values for maternal age, maternal weight and HbA1C in both the groups showed no statistical difference though they have different FBS and RBS levels. Similar groups were taken by Balani for his research.12 When fetal outcome was evaluated it was observed that babies born to mothers on insulin treatment were significantly more in weight as compare to mother given oral metformin. (3.14 kg vs. 3.44, p=0.01). A randomized clinical trial conducted by Niromanesh concluded that babies born to mother given insulin were heavier than metformin treated GDMs.13 Recent study by Marques declared that there were no differences in fetal outcomes in metformin and insulin groups when compared together except neonatal hypoglycemia which was more in the insulin treated group.14 This study was done with much large sample size and a different study design could be the reason for non-similarity in the results of this study with ours. There was no difference for apgar score between both the groups in our study. Similar were the results documented by Rowan upon comparison of neonates of insulin and metformin treated GDMs. He also has found non-significant results for Apgar score at 5 minutes.15 In our study females who received insulin delivered late as compared to females on metformin therapy and the results were statistically significant (p=0.021). Kelley stated that there was significantly less maternal weight gain and lower gestational age at delivery with metformin compared with insulin therapy.16 Whereas Territi stated that there was no difference in both the groups when delivery at gestational age was tabulated.17 There were two intrauterine deaths in insulin groups in our study whereas all the babies were delivered healthy in metformin group confirms that metformin is a safer and effective drug. Rowan stated that 0.2% of females had intra uterine deaths with insulin whereas none of the patient in metformin group had such an issue.15 However results from Hellmuth provide contradictory results to ours, as more intrauterine deaths were observed by him in metformin treated group then in insulin treated group.18 The probable cause could be that females in his study on metformin started treatment in last trimester, when already a level of irreversible hypoxic changes in placenta and fetus had taken place. Carson stated that insulin infusions for several days to the fetal lamb produce a progressive decline in fetal arterial oxygen content and may be one of the factors for more IUDs with insulin treatment.19 With regard to maternal outcomes, it was observed that mean fasting blood sugar at 36 weeks was lowered numerically and HbAIC was significantly less in metformin treated patients as compared to insulin treated GDMs. So we can say that metformin has fulfilled the demands and needs of an anti-glycemic agent. Results by Riaz are in agreement with ours as he documented that metformin for the glycemic control in gestational diabetes mellitus is significantly superior to insulin.20 With regard to mode of delivery, it is clear from our results that surgical deliveries due to Feto-maternal disproportion were in insulin treated patients than in metformin treated patients with a ratio of 7: 2 respectively. These two females in metformin group had surgical deliveries due to narrow pelvic outlet though the babies even in these two cases were not large in size. Goh has highlighted that the caesarean section rate in women with insulin treated GDM was 45.6% due to Feto-maternal disproportion which is much higher than metformin treated group.21 Kale also stated that patients on insulin therapy for GDM have higher rates for cesarean sections and his results are similar to ours.22 Moore discovered that metformin is an effective alternative to insulin as there was no significant difference present in mode of delivery, fetal weight and neonatal complications between insulin treated and metformin treated female in GDMs which is contradictory to our results.23 Waheed also reported that no significant differences were present in mode of delivery and fetal complications, when patients on insulin and metformin treatment for gestational diabetes were compared.3 We also compared the results in both the groups for glycemic values, at the time of enrollment and after the treatment at 36 weeks to evaluate the effectiveness of the treatment. The results showed that metformin decreases the fasting blood glucose levels before and after the treatment significantly (p=0.00). HbAIC was non-significant when compared between the start of study and at term (p=0.061) clearly indicating that metformin has maintained the sugar levels and there was no predicted rise with the advancement of pregnancy. Whereas insulin group showed significant increase in fasting blood sugar levels as well for HbAIC when compared before and after the treatment, (FBS, p=0.002 &HbAIC, p=0.04) indicating that insulin was unable to show expected results. Mesdaghinia declared similar results that not only end term HbAIC but also maternal and fetal complications were much higher in Insulin group and metformin showed better glycemic control and feto-maternal outcome.24 A recent study by Saleh also confirmed the fact that good glycemic control can be achieved using metformin with better fetal and maternal outcomes.25 BODY.CONCLUSION: Metformin has produced better effects on feto-maternal outcomes and glycemic control in patients with gestational diabetes in comparison to insulin. Metformin is used through physiological route, has no storage problem and nor does it requires any dependency. With all these features metformin is a better option than insulin in our population for patients having gestational diabetes mellitus. However multi-centric studies with large sample size are open venues for future research. BODY.CONCLUSION.SOURCE OF FUNDING: Self and Dow University of Health Sciences. BODY.CONCLUSION.WORK PLACE: Dow Diagnostic and Research Laboratory, Dow University of Health Sciences, Karachi, Pakistan. BODY.CONCLUSION.AUTHORS` CONTRIBUTION: RA perceived, designed, conducted research and did main write up of manuscript. SK helped in pathological aspects, write up and editing of manuscript. FS helped in pharmacological aspects, data collection and proof reading of manuscript. NK revised it critically for important intellectual content and is supervisor of the study.

TITLE: Thromboelastographic changes during laparoscopic fundoplication ABSTRACT.INTRODUCTION: Thromboelastography (TEG) is a technique that measures coagulation processes and surveys the properties of a viscoelastic blood clot, from its formation to lysis. ABSTRACT.AIM: To determine the possible hypercoagulability state and the effect of antithrombotic prophylaxis on thromboelastogram results and development of venous thrombosis during laparoscopic fundoplication. ABSTRACT.MATERIAL AND METHODS: The study was performed on 106 patients who were randomized into two groups. The first group received low-molecular-weight heparin (LMWH) 12 h before the operation, and 6 and 30 h after it. The second group received LMWH only 1 h before the laparoscopic fundoplication. The TEG profile was collected before LMWH injection, 1 h after the introduction of the laparoscope and 15 min after the surgery was completed. ABSTRACT.RESULTS: There was no significant difference in thromboelastography R-time between the groups before low-molecular-weight heparin injection. In group I preoperative R-values significantly decreased 1 h after the introduction of the laparoscope, after the end of surgery and on the third postoperative day. K-time values decreased significantly on the third postoperative day compared with the results before low-molecular-weight heparin injection, and after the operation. In group II, preoperative R-values significantly decreased 1 h after the introduction of the laparoscope, and after surgery. K-time values did not change significantly during or after the laparoscopic operation. ABSTRACT.CONCLUSIONS: Our study results demonstrated that the hypercoagulation state (according to the TEG results) was observed during and after laparoscopic fundoplication in patients when LMWH was administered 12 h before the operation together with intraoperative intermittent pneumatic compression. The optimal anticoagulation was obtained when LMWH was administered 1 h before fundoplication. BODY.INTRODUCTION: Venous thromboembolism is an important social and health care problem, because 20–30% of patients develop deep vein thrombosis (DVT) after general surgical operations, while 5.5% of patients have this complication when laparoscopic fundoplication is performed without appropriate prophylaxis [1]. The most frequent reason for pulmonary embolism is thrombi forming in the channels of proximal leg veins and deep pelvis veins. Eighty–ninety percent of pulmonary embolism masses are caused by DVT or a thrombus formed in the pelvis [2]. The development of deep venous thrombosis is related to stasis, hypercoagulability, and injury of the venous wall (Virchow's triad). Laparoscopic surgery causes variable serum hypercoagulability; there are data suggesting that dependent positioning in combination with intraoperative pneumoperitoneum decreases venous flow from the lower extremities and possibly increases the risk of DVT development [3]. The increased intra-abdominal pressure associated with pneumoperitoneum and reverse Trendelenburg position during laparoscopic fundoplication generates venous stasis in the lower limb by compressing the retroperitoneal vena cava and iliac veins, which is already present due to general anesthesia [4]. The intermittent pneumatic compression of the calf with an external pressure cuff for the prevention of DVT is a well-established prevention measure [5–8]. Methods that have conventionally used to prevent postoperative deep vein thrombosis during laparoscopic surgery include not only mechanical techniques, but also drug therapy (low-molecular-weight-heparin – LMWH). Some uncertainties are still present regarding LMWH administration time before elective surgery. Some guidelines suggest LMWH prophylactic administration 12 h before surgery, others 1–2 h before the operation and even 4–6 h after surgery. It is crucial to select the optimal time of LMWH administration in order to obtain the best therapeutic ratio for thrombosis prevention and avoiding excessive bleeding. For this reason, it is important to examine the effect of LMWH on some parameters of coagulation as surrogate markers of possible thrombosis development during and after surgery as it will help to guide physicians with the best time of anticoagulant administration. We chose the thromboelastogram in this respect because it is easy to obtain global coagulation assay with efficacy to detect effects of anticoagulants in various clinical scenarios. Thromboelastography (TEG) analysis is more sensitive to qualitative defects in fibrin or platelets than standard laboratory tests [4]. The technique of thromboelastograph hemostasis analysis provides a measurement of dynamic changes in the viscoelasticity property of the blood clot and gives permanent graphic documentation (Figure 1) [9]; TEG tracing provides all phases of the hemostasis process from the formation of the first fibrin strands to the fibrinolytic dissolution of the clot [4]. Figure 1Characteristic thromboelastographic (TEG) tracings. A – Normal TEG tracing. B – Anticoagulation caused by coagulation factor deficiency or inhibition leading to a prolonged R time. C – Platelet dysfunction or pharmacological inhibition leading to a decreased maximum amplitude (MA). D – Hyperfibrinolysis. E – Hypercoagulability leading to shortened R and K times, with a concomitant elevated and angle BODY.AIM: The aim of this study was to evaluate whether TEG parameters are able to detect the hypercoagulability state in patients undergoing laparoscopic fundoplication and to monitor the effect of the antithrombotic prophylaxis before, during and after laparoscopic fundoplication. BODY.MATERIAL AND METHODS: This was a prospective randomized clinical study, where 106 patients undergoing elective laparoscopic fundoplication because of gastroesophageal reflux disease, caused by hiatal hernia, were studied. All the patients gave their written informed consent, and The Kaunas Regional Biomedical Research Ethics Committee approved the study (protocol no. BE-2-13). This randomized clinical study was also registered on the ISRCTN registry with trial ID ISRCTN62203940. All the patients were randomized into two groups (Figure 2). The first group of 55 patients received LMWH Bemiparinum (Zibor, Berlin Chemie, Luxembourg) 2500TV 0.2 ml subcutaneously 12 h before the operation, and 6 and 30 h after it. The second group of 51 patients received LMWH Bemiparinum 2500 TV 0.2 ml. subcutaneously 1 h before the operation. Both groups received intermittent pneumatic compression (IPC) during all laparoscopic fundoplication. The IPC was performed using "Kendall SCD 700 Series" apparatus. We acquired 5-mm-thick axial CT (GE Light Speed Pro) venograms from the ankle to the iliac wing after injection of 150 ml of 300 mg/ml contrast medium at a flow rate of 3 ml/s through an antecubital vein on the third postoperative day. Figure 2Study design BODY.MATERIAL AND METHODS.ANESTHESIA: All operations were performed under standardized endotracheal anesthesia. Premedication: all patients received 7.5 mg of midazolam 1 h before transfer to the operating room. In the operating room standard monitoring was established including an electrocardiogram (five electrode system), peripheral oxygen saturation, and non-invasive blood pressure ("S/5 Compact" Datex-Ohmeda). Preoxygenation with 100% oxygen via a face mask for 3 min was followed by standardized induction of anesthesia with thiopentone 4–6 mg/kg, fentanyl 2.5 μg/kg, and pipecuronium 0.08 mg/kg, and anesthesia was subsequently maintained with isoflurane 0.6–0.7 of minimal alveolar concentration and fentanyl 1.0–1.25 μg/kg every 20–25 min. Lungs were artificially ventilated with "S/5 Aespire" Datex-Ohmeda (FiO2 0.40–0.45), respiration was monitored with "S/5 Compact" Datex-Ohmeda, and parameters of artificial lung ventilation were changed according to readings of capnography, keeping end tidal partial pressure of CO2 at 35–40 mm Hg. Neuromuscular blockade was reversed by atropine 1.0 mg and neostigmine 2.0 mg. All patients were extubated in the operating room. Infusion therapy: before insufflation all patients received intravenously 500 ml of Ringer's solution; the total amount of fluid during the operation was 1500 ml of Ringer's solution. BODY.MATERIAL AND METHODS.OPERATION: Two experienced surgeons performed all the operations. Pneumoperitoneum was induced by inserting a Veress needle above the umbilicus and connecting it to the CO2 insufflator, which achieved and maintained an intraabdominal pressure of 12–14 mm Hg. The patients were moved to the reverse Trendelenburg position (45°) before intraabdominal manipulation. Five trocars were inserted into the peritoneal cavity at the typical sites – at the epigastrium, in the right subcostal area, two in the left subcostal area, and one on the middle abdominal line above the navel. The short gastric arteries were divided; the same procedure was performed on the gastric diaphragm ligament, releasing the gastric fundus. On the side of the small gastric curve, the liver-stomach ligament was cut, and the right diaphragm leg was prepared, followed by the left one. The mobilization of the esophagus was completed through bypassing it in the circular manner, freeing a 3–4 cm-long intraabdominal segment and removing the hernia sack. The diaphragm legs were then sutured using a 3–4-suture non-resorbing Ethibond (Ethicon, Johnson & Johnson Company, USA) behind the esophagus. The next step of surgery was the formation of a 360° (Nissen) antireflux wrap. It was formed by rolling a 2.5 cm long wrap of gastric fundus behind the abdominal portion of the esophagus, thus reinforcing the lower esophageal sphincter. The wrap was then fixed to the esophagus and the stomach was sutured to the front of the esophagus. BODY.MATERIAL AND METHODS.BLOOD COLLECTION AND PROCESSING: Venous blood samples for TEG analysis were taken from the forearm vein four times: before LMWH injection an before the operation;1 h after the introduction of the laparoscope;15 min after extubation;on the third postoperative day. Blood was drawn by a clean venipuncture following a double-syringe technique and placed into vacutainer tubes containing 3.9% (0.129 M) trisodium citrate (Becton Dickinson Vacutainer Systems, Plymouth, UK) in the ratio of one part of citrate to nine parts of whole blood. Whole blood was transferred within 5 min to prewarmed cuvettes of the thromboelastograph. The TEG analysis was performed with TEG 5000 Thromboelastograph Hemostasis Analyzer system (Haemonetics Corporation, UK). The coagulation process was activated using Kaolin and 20 μl of calcium chloride as described in the literature. The TEG system's approach to the monitoring of patient hemostasis is based on the premise that the end result of the hemostasis process is a single product, which is the clot [4]. The TEG system measures the clot's physical properties by the use of a cylindrical cup that holds the 0.36 ml blood as it oscillates through an angle of 4°45' and each rotation lasts for 10 s (Figure 3). As the clot begins to form in the presence of the activator, the TEG records the visceroelastic changes that occur in the sample. Figure 3Technique to perform the TEG test A pin suspended in the blood within a stationary cup is monitored for motion as fibrin-platelet bonding occurs. This motion is related to the strength of the clot and is analyzed by a computer, which creates a graphical representation. The TEG is a point-of-care whole blood coagulation monitor which provides information on specific aspects of coagulation including time to production of initial fibrin strands (R-time), time to develop a clot (R-time, K-time), rate of fibrin build-up and cross linking (a-angle), maximum clot strength (maximum amplitude – bMA) and measures of fibrinolysis (decreasing amplitude post-MA) [10] (Figure 4). Figure 4TEG tracing – normal values of TEG parameters (celite activated whole blood)R-time – reaction time, in minutes (norm: 5.0–10.0 min) – this denotes the time taken from the beginning placement of the blood sample in the cuvette to the initial fibrin formation. K-time – clotting time (norm; 1.0–3.0 min) representing the time of fixed clot formation. &-angle – degrees; closely related to K (norm: 53.0– 72.0°) this represents the rate of clot growth. MA parameter – maximum amplitude, millimeters (norm: 50.0–70.0 mm) – a measurement of maximum strength of the developed clot. The thromboelastographic trace was analyzed for: R-time (reaction time, in minutes) – it is time from the beginning of the trace until an amplitude of 2 mm is reached. The period of latency time is taken from the placement of the blood sample in the cuvette to the initial fibrin formation.K-time (clotting time, in minutes) – it is the time from the end of R until a fixed clot strength is reached, i.e. amplitude of the trace is 20 mm.&-angle (clot formation rate) – it is affected primarily by the rate of thrombin generation.MA (maximum amplitude) – it is a measure of maximum strength of the clot and assesses the function of the platelets and to some extent fibrinogen also. BODY.MATERIAL AND METHODS.DEEP VEIN THROMBOSIS DIAGNOSIS: All the patients underwent color duplex scan examination preoperatively and spiral CT venography with color duplex scan on the third postoperative day in order to detect possible DVT. One experienced radiologist performed all these examinations. Criteria for DVT diagnosis were: intraluminal filling defect or localized nonopacification of the venous segment. BODY.MATERIAL AND METHODS.STATISTICAL ANALYSIS: Statistical evaluation was conducted using descriptive analysis: normality test (Shapiro-Wilk W-test), the unpaired Mann-Whitney U test to compare data from two study groups, and the Wilcoxon test to investigate differences in TEG values in each group of patients. Data are expressed as mean ± SD. P-value < 0.05 was considered significant. BODY.RESULTS: The patients in both groups were similar in terms of age, weight, height, gender, duration of surgery, and American Society of Anesthesiologists (ASA) class (Table I). There was no massive or minor bleeding during all laparoscopic operations. No drains were left after the operation. Computed tomography venography revealed popliteal vein thrombosis in 2 (3.6%) group I patients on the third postoperative day (Photo 1). All the patients left the hospital after an uneventful 3–5-day stay. Table I Patients’ demographic characteristics Value Group I ( N = 55) Group II ( N = 51) P -value Age [years] 55.27 ±14.25 55.24 ±14.65 0.886 Male/female ( n / n ) 18/37 16/35 0.881 BMI [kg/m 2 ] 27.03 ±5.04 26.72 ±4.82 0.463 Duration of surgery [min] 136.73 ±45.16 129.71 ±36.84 0.638 ASA class:  I 7 6 0.921  II 29 30 0.921  III 18 15 0.921  IV 1 0 0.661 Postoperative stay [days] 3.98 ±0.913 4.04 ±0.979 0.835 Values are mean ± SD. Photo 1Computed tomography venography revealed v. poplitea dex. thrombosis: A – transverse CT scan, B – coronal CT scan TEG values – R-time, K-time, α-angle and maximum amplitude – were not significantly different between the groups before LMWH injection, during and after laparoscopic fundoplication (Tables II–V). Table II R-time value changes in both groups Study groups Before LMWH injection 1 h after introduction of the laparoscope After extubation Third postoperative day I ( N = 55) 7.41 ±1.51 ♦ ■ ● 6.40 ±2.65 ♦ 6.28 ±1.90 ■ 6.09 ±1.44 ● II ( N = 51) 6.36 ±1.84 * ∞ 5.10 ±1.64 * 5.35 ±2.15 ∞ 5.87 ±2.12 ♦ p = 0.002 ■ p < 0.001 ● p < 0.001 * p = 0.010 ∞ p = 0.001. R-time (5.0–10.0 min). Table III K-time value changes in both groups Study groups Before LMWH injection 1 h after introduction of the laparoscope After extubation Third postoperative day I ( N = 55) 2.68 ±1.50 ♦ 2.59 ±1.97 2.82 ±1.61 ■ 2.22 ±1.04 ♦ ■ II ( N = 51) 2.13 ±1.29 2.01 ±1.57 2.12 ±2.25 2.14 ±1.71 ♦ p = 0.020 ■ p = 0.002. K-time (1.0–3.0 min). Table IV Alpha-angle value changes in both groups Study groups Before LMWH injection 1 h after introduction of the laparoscope After extubation Third postoperative day I ( N = 55) 55.12 ±11.35 55.88 ±14.02 54.93 ±13.91 57.25 ±12.14 II ( N = 51) 59.34 ±13.81 ■ ● 65.24 ±12.68 ■ 64.71 ±15.01 ● 62.35 ±15.12 ■ p = 0.010 ● p = 0.012. Alpha-angle (53.0–72.0°). Table V Maximum-amplitude value changes in both groups Study groups Before LMWH injection 1 h after introduction of the laparoscope After extubation Third postoperative day I ( N = 55) 64.86 ±8.97 66.13 ±9.33 63.17 ±8.20 ♦ 65.98 ±7.29 ♦ II ( N = 51) 63.13 ±11.85 ■ 61.11 ±22.29 64.41 ±7.21 66.20 ±7.03 ■ ♦ p = 0.015 ■ p = 0.028. Max. amplitude (50.0–70.0 mm). In group I, preoperative TEG R-values significantly decreased already 1 h after the introduction of the laparoscope, and remained at the same level until the third postoperative day (Table II). TEG K-time values decreased significantly only on the third postoperative day compared with the results before LMWH injection, during the surgery or after the operation (Table III). In group II, TEG R-time and K-time values did not change significantly during or after the laparoscopic operation (Tables II and III). There were no significant changes in α-angle and maximum amplitude values during or after laparoscopic fundoplication in both groups (Tables IV and V). BODY.DISCUSSION: Thromboelastography is a viscoelastic hemostatic assay that measures the global visco-elastic properties of whole blood clot formation under low shear stress. Thromboelastography also is a method to monitor the hemostasis that is easy to use in the operation theatre, and the anesthesiologist is provided with rapid and accurate results. The formation of the clot is a net result of interaction between the cellular components of blood and coagulation proteins. The overall coagulation profile can be quantitatively and qualitatively interpreted in terms of the hypocoagulable, normal or hypercoagulable state of the sample and degree of lysis [11]. The TEG is a technique that gives a global overview of the coagulation cascade including fibrinolysis. Many workers have tried to correlate TEG parameters with the conventional coagulation profile, but it is not possible as the two techniques are different [11]. Commonly used blood tests are often aspecific, whereas the dosage of all plasma factors involved in coagulation and platelet activity is expensive and mostly not useful to individuate the clinical risk of hypercoagulability [12]. But to some extent, R correlates with activated partial thromboplastin time (APTT), and angle correlates with fibrinogen (and platelet function), MA correlates with platelet function (and fibrinogen) [13–18], and whole blood clot lysis index correlates with euglobulin lysis time and fibrin degradation products (FDP) [11]. It is already proven that laparoscopic surgery leads to postoperative activation of the coagulation system, which is a prerequisite for thromboembolic complications. Many factors can cause postoperative changes of hemostasis. These include the type of operation performed and the type of anesthesia, duration of the operation, and transfusion [19]. In laparoscopic surgery, the pneumoperitoneum reduces venous flow from the lower extremities by approximately 30% to 40% [19]. Therefore, the risk of DVT might be increased after laparoscopic surgery compared with open surgery. Topal et al. [19] showed that high intra-abdominal pressure led to a significant variation in hemostasis and coagulation compared with low intra-abdominal pressure. They used 10, 13 and 16 mm Hg intra-abdominal pressure with the standard anesthetic technique in this study. Conventional tests only monitor a single point of LMWH's effect on the coagulation cascade, whilst TEG can be used to assess the full anticoagulant effects of LMWH [20]. The LMWHs are unique in their ability to preferentially inhibit factor Xa more than factor IIa. Low-molecular-weight heparin has multiple potential effects on the coagulation pathway including antifactors IIa, IXa and Xa activity, inhibition of thrombin-induced activation of factors V and VIII, platelet function inhibition, and enhanced fibrinolysis and endothelial secretion of tissue factor pathway inhibitor [20–22]. Klein et al. [22] reported that thromboelastographic R value correlates significantly with the peak and trough levels of anti-Xa activity after the administration of LMWH. Anti-factor Xa activity is not advised as a routine screening test as it does not reliably predict the risk of peri-operative bleeding [23, 24]. Our study compared the occurrence of DVT and thromboelastometric parameter changes during and after laparoscopic fundoplication with different pharmacologic prevention: low-molecular-weight heparin administered 1 h or 12 h before the surgery. Bemiparin is a LMWH indicated for the acute treatment of deep vein thrombosis with or without pulmonary embolism, for the prophylaxis of venous thromboembolism in surgical and nonsurgical patients and for the prevention of clotting in the extracorporeal circuit during hemodialysis [25]. Bemiparin is the second generation LMWH with the lowest molecular weight, the longest half-life [16] and the highest anti-factor Xa/anti-factor IIa activity ratio. Our study results demonstrated that the low-molecular-weight heparin protected from possible operation-induced hypercoagulation when it was administered 1 h before laparoscopic fundoplication – we found no difference in R- and K-time, MA, and α-angle compared with baseline values in thromboelastographic examination. Despite the IPC action, when LMWH was administered 12 h before surgery, significant intraoperative and postoperative shortening of R time and K time on the third postoperative day and no difference in MA and a-angle were detected. These findings suggests that the hypercoagulability state is present, which starts already during laparoscopic fundoplication and continues some days after it. These results of ours nicely correspond with LMWH pharmacokinetic data: bemiparin is rapidly absorbed after subcutaneous administration, attaining maximal plasma anti-Xa activity within 2–6 h. Postoperative administration of bemiparin (6 and 30 h after fundoplication) does not protect from intervention-induced hypercoagulation as R- and K-time in this patient group remained shortened even on the third postoperative day. These results of ours demonstrate that prevention of operation-induced hypercoagulability is of crucial importance and it can be achieved with timely LMWH administration (one hour before laparoscopic surgery). Caprini et al. [26] investigated TEG preoperatively and on the day following the operation in 100 laparoscopic cholecystectomy cases. They found significant postoperative shortening of R and K but no difference in MA. They suggested that postoperative coagulation asthenia should be attributed not to platelets but to coagulation activation. This was considered to reflex excessive enhancement of the activation of coagulation factors. Forfori et al. [12] analyzed the correlation between the variation of normal TEG parameters and the dosage amount of anticoagulant therapy adjusted to body weight. They found no correlation between the variations of all screened TEG parameters from basal to postoperative and pro/kilogram dosage of LMWH administered. The authors suggest that LMWH prophylaxis based on weight produces a feasible but not a more efficient anticoagulation profile than standard LMWH therapy. The exact incidence of thromboembolic complications after laparoscopic fundoplication is unknown, and we could not find studies on the dynamics of intraoperative and postoperative coagulation during laparoscopic fundoplications. Our study identified two cases of calf deep vein thrombosis (3.6%) only in the group in which LMWH was administered 12 h before laparoscopic surgery. Keeping in mind that patients after laparoscopic fundoplication are mobile within a few hours after surgery, we can speculate that calf deep vein thrombosis in these two patients occurred during the operation. According our study results, we can also assume with caution that prophylaxis with intraoperative IPC and LMWH 12 h before surgery is not efficient enough to protect against the hypercoagulation state, present during laparoscopic fundoplication. On the other hand, the main weakness of our study is that the number of patients is too small in both groups, so it is impossible to find a significant difference in the rate of DVT between the groups. Venous stasis in laparoscopic operations can be minimized by reducing intraabdominal pressure during the operation. Combination of LMWH 1 h before laparoscopic surgery and intraoperative IPC generates a hypocoagulation effect more effective to prevent deep-vein thrombosis during or after the laparoscopic operation. BODY.CONCLUSIONS: Monitoring TEG parameters can be a feasible practice to observe the coagulation status in patients undergoing laparoscopic surgery. Our first group patients presented a tendency to the hypercoagulability state, determined mostly by the shortening of TEG R-time. Comparison of TEG values between the study groups indicates that prophylaxis with LMWH 1 h before surgery together with IPC protects patients from surgery-induced hypercoagulation, and this lasts at least for three days. Our recommendation is that LMWH, as a DVT prophylactic measure, has to be administered 1 h before the laparoscopic operation to ensure the drug's optimal effect.

TITLE: Effect of acupressure on fatigue in patients on hemodialysis ABSTRACT.BACKGROUND:: Fatigue is considered as a major problem in hemodialysis patients and can impair their quality of life. The purpose of this study was to investigate the effectiveness of acupressure on fatigue in hemodialysis patients. ABSTRACT.MATERIALS AND METHODS:: This is a clinical trial study in which 96 hemodialysis patients participated. Patients were randomly assigned into acupressure, placebo, and control groups (32 subjects fulfilling the inclusion criteria assigned to each group). The measures included the form of demographic characteristics, visual analog scale of fatigue, and Piper Fatigue Scale. Patients in the acupressure and placebo groups received acupressure intervention during the early 2 h of dialysis on six acupoints with massage for 20 min/day, 3 days per week for 4 weeks. In the placebo group, acupressure intervention was performed as mentioned above with a distance of 1 cm away from the actual intervention site. Patients in the control group received routine unit care only. Chi- quare test, Kruskal-Wallis, paired t-test, one-way analysis of variance (ANOVA), and Duncan test were used for data analysis. ABSTRACT.RESULTS:: One-way ANOVA tests showed significant differences in the total mean score of fatigue and fatigue mean scores in the behavioral, emotional, sensory, and cognitive dimensions in the acupressure, placebo, and control groups. ABSTRACT.CONCLUSION:: The results of this study showed that acupressure may reduce fatigue in hemodialysis patients, and use of this non-pharmacologic technique for hemodialysis nurses is suggested. BODY.I: One of the very common chronic diseases is chronic renal failure,[1] which may lead to end-stage renal disease (ESRD) as a part of that.[2] Its yearly incidence has been reported 330 cases out of 1 million in the US and 253 cases out of 1 million people in Iran. Based on the last statistics in 2007, there are 16,600 hemodialysis patients in Iran.[3] ESRD patients need renal replacement therapy to survive,[4] and hemodialysis is the most prevalent conventional renal replacement therapy in Iran.[5] ESRD patients undergoing chronic hemodialysis suffer from numerous complications including fatigue.[6] The range of fatigue prevalence has been reported to be 60%-97% among hemodialysis patients.[7] This sign is a complicated concept and its diagnosis and assessment is difficult for the nurses.[8] In ESRD patients, untreated fatigue may highly affect the quality of life and lead to patients' increased dependency on others, weakness, loss of physical and psychological energy, social isolation, and depression. The elements that can affect the level of fatigue include depression, anemia, sleep disorders, and restless leg syndrome.[9] Medicational interventions such as consumption of growth hormone, anti-depressant and anti-anxiety medications, and levocarnitine and erythropoietin stimulating hormone, as well as non-medicational interventions such as nutrition therapy, sleep disorder treatment, stress management, sport, Yoga, depression treatment, drug abuse, and acupressure are used to lower hemodialysis patients' fatigue.[7] Because of medications' side effects and incomplete relief of fatigue after taking anti-fatigue medications, the patients are driven to complementary medicine methods such as acupressure medicine.[10] Based on the reported researches, acupressure medicine is one of the interventions with the highest application by nurses in clinical settings,[11] and is considered as a clinical and comprehensive nursing intervention.[12] This is also among the manual treatments for which the nurses have an excellent position to use.[13] Acupressure medicine, as a healing art, is beautifully integrated with nursing[14] as it is non-invasive and has advantages such as cost efficacy, lack of complications, no need for special tools, and the ease of learning for the patients and their accompanying persons.[12] The only study conducted on hemodialysis patients' fatigue was Cho and Tsay's study (2004) in Taiwan, which showed that acupressure medicine can lower the level of fatigue in patients of experimental group compared to control group.[8] Nasiri et al. (2007) in a study on the association between the effect of acupressure medicine and sleep quality of hemodialysis patients showed a significant difference in the sleep quality of experimental and control groups based on Pittsburgh Sleep Scale.[3] Previous studies include a systematic review on the effect of acupressure medicine on hemodialysis patients' signs including fatigue,[15] and meta-analysis studies on the effect of acupressure medicine on labor pain management in labor unit[16] as well as its effect on post-operation vomiting and nausea in adults.[17] Their results showed that despite poor methodology, high risk of bias, and low number of subjects, it cannot be absolutely indicated that acupressure medicine is effective on patient's signs. Tsay (2004) in a study on the effect of acupressure medicine on hemodialysis patients' fatigue stated that with regard to cultural and lifestyle differences of the patients in various countries, the results of these studies cannot be generalized for the patients in other geographic areas.[18] Therefore, with regard to the difference between the Iranian culture and the culture in other countries,[19] and as no study has been conducted on the effect of acupressure medicine on hemodialysis patients' fatigue on the one hand and due to the high application of acupressure medicine in clinical nursing interventions on the other hand (as this type of medicine can provide the patients with a cost-effective and safe nursing intervention), this study was conducted as there was no need of any special tools but just trained finger tips and it could be conducted within a shorter time. Therefore, the researcher decided to conduct a study with the goal of defining the effect of acupressure on hemodialysis patients' fatigue. BODY.M: This is a three-group clinical trial. Study population included all ESRD patients undergoing chronic hemodialysis in three hemodialysis centers of Nour, Alzahra, and Shariati, hospitals. The subjects were selected from the study population based on the inclusion criteria. Inclusion criteria were age of over 18, diagnosis of ERSD, undergoing hemodialysis at least for 3 months, the patients with chief complaint of fatigue and having fatigue score ≥5 based on fatigue severity visual analogue scale, lack of any wound or fracture, being in complete psychological and mental health to attend the study and fill the questionnaire, and not having undergone complementary medicine treatment in the past 3 months of the study. Exclusion criteria were patient's absence for two sessions of acupressure intervention and lack of interest to continue the study. The sampling was convenient continuous sampling. The number of the subjects was estimated to be 96. After random subjects' allocation through minimization method, 32 subjects were assigned to each group of the study, placebo and control. The data were collected by a three-section questionnaire whose first section contained demographic characteristics including age, sex, education level, marital status, occupation status, and disease etiology. The second section contained Fatigue Severity Scale (FSS) with a visual analogue scale ranked between 0 and 10, which was scored and commented as fatigue (0), minor fatigue (1-3), moderate fatigue (4-6), and severe fatigue (7-10). If the patients had fatigue severity score of ≥5, they attended the study. FSS is a standard scale whose validity and reliability have been confirmed by Gift,[20] and has been frequently used in various studies. The third section included Piper Fatigue Scale with 27 items on four dimensions of mental fatigue including behavioral, emotional, sensory, and cognitive dimensions. Each item was scored from 0 to 10, with score zero given for lack of fatigue, 1-3 for minor fatigue, 4-6 for moderate fatigue, and 7-10 for severe fatigue. Piper Fatigue Scale is a standard scale whose validity (content and concurrent) and reliability have been confirmed. In the study of Masoodi et al.,[21] its scientific validity was confirmed by the professors of Tarbiat Modares, Tehran, and Iran universities. Its reliability was confirmed in the study of Masoodi et al. by a correlation coefficient of r = 0.89. In the present study, its internal validity was α = 0.77 and its reliability was calculated as 81.2% by test retest. This scale was distributed among the patients once before the intervention and once after the intervention to score the subjects' fatigue severity. Intervention was conducted in the first 2 h of hemodialysis in the experimental and placebo groups. This intervention was carried out among the subjects regularly undergoing hemodialysis, in both legs, hands, and the waist in three weekly sessions for 4 weeks.[18] In the experimental group, the intervention was conducted on the major acupoints K1, GB 34, ST 36, SP 6, BL 23, and HT7.[2223] In the placebo group, it was carried out with 1 cm distance from the major above-mentioned acupoints. Each session lasted for 20 min, of which 2 min were devoted for primary superficial stroking of the acupoints[24] and the rest of the time (18 min) was for acupressure of the determined six acupoints (3 min for each acupoint).[25] The researcher and a male co-researcher were trained under the supervision of the second supervisor, and then the intervention started by her approval. The researcher (female) and her male co-researcher conducted the intervention separately for the female and male subjects, respectively. Determination of acupoints was made based on the second supervisor's guidance on the acupoints' standard location. The amount of needed pressure was practiced by standard scales of 20 g to 6 kg, and the reliability of pressure level was confirmed as 100% after 40 times of practice with mean of 3-4 kg on the scales for both researchers' hands. The details about the process of research, goals, and conditions were given to the patients, and consequently, those interested to join entered the study after filling the written consent form. Sampling and intervention were conducted after obtaining approval from ethical consideration committee of Nursing and Midwifery School of Isfahan University of Medical Sciences. The data were analyzed by chi-square, Kruskal-Wallis, paired t-test, one-way analysis of variance (ANOVA), and Duncan's test through SPSS version 19. BODY.R: Mean age of the subjects in the experimental group was 53.4 (13.9) years, in the placebo group 55.4 (11.5) years, and in the control group 54.3 (13.4) years. In the experimental, placebo, and control groups, there were 14 female (43.75%) and 18 male (56.25%) subjects, respectively. The most prevalent etiology of the disease was diabetes in 11 subjects (34.4%) in the study group, hypertension in 14 subjects (43.8%) in the placebo group, and diabetes in 12 subjects (37.5%) in the control group. Before intervention, subjects' matching was conducted concerning demographic characteristics in the three groups of study, placebo, and control through minimization program, and there was no significant difference in the demographic characteristics between the groups (P > 0.05). One-way ANOVA showed that there was no significant difference between the total mean score of fatigue and the mean scores of Piper Fatigue Scale dimensions before intervention in all the three groups, but these means showed a significant difference after intervention in the three groups (P < 0.001). Post-hoc Duncan test showed that in the study group, total score of fatigue and score of fatigue in emotional, cognitive, and behavioral dimensions were significantly less than in the two groups of placebo and control after intervention (P < 0.05), but there was no significant difference between the control and placebo groups (P > 0.05). Mean score of fatigue in the sensory dimension in the study group was significantly less than in the placebo group (P < 0.05), and in the placebo group, it was significantly less that in the control group (P < 0.05). As observed in Table 1, the total mean score of fatigue and the mean scores of fatigue in sensory, behavioral, cognitive, and emotional dimensions in the two groups of study and placebo were less after intervention compared to before intervention, and paired t-test showed a significant difference (P < 0.05). The total mean score of fatigue and the mean score of fatigue in sensory, behavioral, cognitive, and emotional dimensions showed no significant difference before and after intervention (P > 0.05). As observed in Table 2, there was a significant difference in the total mean score change of fatigue and the mean score change of fatigue severity in all dimensions in the three groups after intervention (P < 0.001). Post-hoc Duncan test showed that reduction of mean fatigue score in behavioral and emotional dimensions was more in the study group compared to the other two groups of placebo and control (P < 0.05), but it showed no significant difference in the two groups of control and placebo (P = 0.3). Post-hoc Duncan test showed that mean reduction of total fatigue score and mean reduction of fatigue scores in sensory and cognitive dimensions in the study group were more than in the placebo group, and they were more in the placebo group compared to the control group (P < 0.05). Table 1 Comparison of total mean scores of fatigue, behavioral, emotional, sensory, and cognitive dimensions before and after intervention in the three groups of study, placebo, and control Table 2 Comparison of fatigue score changes and dimensions of piper fatigue scale after intervention in the three groups of study, placebo, and control BODY.D: The results of acupressure medicine on severity of fatigue in hemodialysis patients showed that the study group experienced less fatigue severity compared to the placebo and control groups. As the subjects' mean age in the present study was over 50 years, it is concluded that ESRD seems to be more prevalent at higher ages. Tsay and Chen (2003) reported more ESRD prevalence at higher ages.[26] The higher number of male subjects in the present study reveal that ESRD prevalence seems to be more prevalent among men, which concords with the findings of Nasiri et al.[3] Most of the subjects were retired or jobless in the present study. Studies show that the rate of joblessness is high among hemodialysis patients.[27] Diabetes and hypertension are the major etiologies for ESRD,[18] which is consistent with the finding of the present study. Stimulation of acupoints results in improvement of vital energy circulation in the body and increase of opioids like endorphin and enkephalin, which may play a role in the reduction of patients' fatigue in all behavioral, sensory, cognitive, and emotional dimensions.[18] The obtained results showed that there was a significant reduction in the total mean score of fatigue in the study group after intervention compared to the placebo and control groups. Although a similar improvement was observed in the placebo group based on Duncan test, the improvement was more in the study group. It seems that in the placebo group, in addition to verbal and psychological communication between the researcher and the patients, due to the physiologic and psychological effects of acupressure, the patients expect the acupressure to have a positive effect on their body, named as Hawthorne phenomenon. So et al. (2007) in a study aimed to define the effect of Transcutaneous Electrical Acupoint Stimulation (TEAS) on improvement of muscular fatigue among athletes and showed a significant difference between TEAS and placebo groups in return of muscular power.[28] This study is in line with the present study except for the lack of a significant difference in the placebo groups after intervention. The difference between these two studies seems to be as a result of the study population, intervention method and the number of subjects. The results showed that the mean score of fatigue in emotional dimension significantly diminished in the study group after intervention compared to the placebo and control groups. From psychological aspect, acupressure induces alpha brain wave stimulation that consequently leads to relaxation and reduction of fatigue and anxiety.[28] This medicine has a positive effect on the body and mind and brings about mental joyfulness and health through stimulation of opioid — endogen systems such as endorphin and enkephalin.[29] Molassiotis et al. (2007)[30] reported the significant effect of acupuncture and acupressure on cancer patients' fatigue in three groups of acupressure, acupuncture, and placebo in the dimensions of general fatigue, activity, and physical fatigue, although it had no significant effect on the dimension of motivation and psychological fatigue. The difference, observed even in the control group, seems to be as a result of the effect of researcher's psychological factors. Their results showed that acupuncture was more effective than acupressure and acupressure was more effective than placebo. This finding is not consistent with our study, possibly due to the difference in the study population, the scale used, and the length of intervention. Our obtained results showed a significant reduction in the mean score of fatigue in sensory dimension in the study group after intervention compared to placebo and control groups. The study results of Tsay et al.[29] and the results of the present study are consistent due to use of similar acupoints and length of intervention. The only difference is that there was no placebo group in their study, and in the control group, the mean score of sensory dimension had a reduction, possibly as a result of psychological and mental elements. Our results showed a significant reduction in the mean score of fatigue in cognitive dimension in the study group after intervention compared to the placebo and control groups, which can be due to the effects to acupressure mechanism and researcher's psychological and mental factors during the study. In the study of Harris et al.[31] in which the effect of the two methods of stimulating acupressure medicine and relaxation on students' consciousness and fatigue was studied, stimulating acupressure significantly diminished students' fatigue and increased their daytime consciousness. Their consistent results with the present study may be due to the effect of acupressure mechanism, while there was no difference in the placebo group in this study. Our results showed a significant reduction in the mean score of fatigue in behavioral dimension after intervention in the study group compared to the placebo and control groups, as the acupoints used in the present study could have had a synergic effect to reduce fatigue. Hosseinabadi et al.[32] reported that acupressure was effective on the daily function of Pittsburgh Sleep Quality Scale in the elderly in the study group, and there was a significant difference between the study and placebo groups, which is not consistent with our study possibly due to the difference in study population. The results showed a significant reduction in the mean score of fatigue severity in the study group after intervention compared to the placebo and control groups. In a study of Molassiotis et al.,[30] the mean reduction score of fatigue severity in acupuncture group was more than in acupressure and placebo groups, which is not consistent with the results of the present study. This difference seems to be as a result of the type of intervention, length of time, frequency of intervention, the acupoints used, and/or the measurement scale used. Based on most of the experts' ideas, sham acupoints also have a treatment effect as most of the time, positive effects also occur in the placebo groups.[33] With regard to the inefficiency of the used acupoint in the placebo group, patients' reduction of signs can be as a result of their expectation concerning a positive effect from the intervention domination and/or they felt secure by the presence of the researcher as well as Hawthorne effect. BODY.C: The results of the present study with regard to consideration of fatigue as a problem in hemodialysis patients and application of acupressure medicine by nurses in most of their clinical interventions showed that this non-invasive, cost-effective, learnable, and complication-free method can be used to lower the fatigue in hemodialysis patients. Therefore, this caring method of complementary medicine is suggested to be used by nurses in hemodialysis wards to lower the patients' fatigue.

TITLE: Managing migraine headaches experienced by patients who self–report with menstrually related migraine: a prospective, placebo–controlled study with oral sumatriptan ABSTRACT: The objective was to evaluate the efficacy and tolerability of oral sumatriptan (100 mg) in patients who self–reported with menstrually related migraine. A prospective, multicentre, randomised, double–blind, placebocontrolled, two–group crossover study was carried out in 20 UK primary and secondary care surgeries. Of 115 patients with a self–reported history of menstrually related migraine that entered the study, 93 patients completed it. Patients treated all migraine attacks for 2 months with sumatriptan (100 mg) and for 2 months with placebo. The primary endpoint was the proportion of patients reporting headache relief at 4 hours for the first treated attack. Only 11% of patients fulfilled the protocol definition of menstrually related migraine. Patients reported a variable pattern of migraine attacks occurring inside and outside the menstrual window. For the first attack, significantly more patients receiving sumatriptan than placebo reported headache relief for attacks occurring inside (67% vs. 33%, p=0.007) and outside (79% vs. 31%, p<0.001) the menstrual period. Sumatriptan was generally well tolerated. Oral sumatriptan (100 mg) is an effective and well tolerated acute treatment for patients who report menstrually related migraine.

TITLE: Impact of handwriting training on fluency, spelling and text quality among third graders ABSTRACT: As recent studies and theoretical assumptions suggest that the quality of texts composed by children and adolescents is affected by their transcription skills, this experimental field trial aims at investigating the impact of combined handwriting/spelling training on fluency, spelling and text quality among normally developing 3rd graders (N = 175). In addition to the combined handwriting/spelling training group, the sample includes two other intervention groups, a handwriting training group and a spelling training group as well as a reading fluency training control group. The participating teachers (N = 11) and their students were randomly assigned to the different intervention and control conditions, which were scheduled to last 20 units (each unit lasts 15 min) distributed over 5 weeks (4 units/week). Data collection was administered both before (pre-test) and after (post-test) the intervention as well as 3 months later (follow-up). Measures included group-administered tests and tasks (spelling, visuo-motor integration, copy task, and composing) and individually administered tests and tasks (working memory and several handwriting tasks on the digitizing tablet). As handwriting automaticity (measured on the digitizing tablet) was already high at the beginning of the study, the intervention was not able to improve it further. In consequence, an intervention effect on the quality of composed texts was not observed. Instead, text quality was predicted by working memory, fluency, spelling, and gender irrespective of the intervention. BODY.INTRODUCTION: Handwriting is a cultural skill and is taught and used in every school around the world, as well as applied in leisure activities and labour contexts later on despite the fact that keyboard typing is recently becoming more and more prevalent early in life. BODY.INTRODUCTION.MODELS OF HANDWRITING: Most scholars understand handwriting as a complex neuromotor skill involving cognitive and motor processes sequentially as well as simultaneously. Contemporary models assume that these processes are controlled by a hierarchic architecture of both central and peripheral components (Kandel, Peereman, & Ghimenton, 2013) operating partially in a parallel mode (e.g. Kandel, Peereman, Grosjacques, & Fayol, 2011; Van Galen, 1991; Van Galen, Meulenbroek, & Hylkema, 1986). According to Hayes (2012a, b), composing (handwriting as well as typing) consists of higher and lower level processes whereby handwriting is conceptualized within the low-level transcription process that follows planning and translating. While higher-level processes include ideation, choice of words (semantic coding) and syntax, lower-level processes refer to the retrieval of orthographic and allographic representations as well as the activation of neuromuscular networks and the subsequent execution of fine-motor movements. In general, higher order processes precede lower order processes, however, at least some processes are supposed to be synchronic, i.e. a writer executes an allograph while planning to write the next syllable during continuous handwriting (Kandel, Álvarez, & Vallée, 2006; Kandel et al., 2011). In addition, recent research indicates that some central processes are still ongoing during movement execution, i.e. showing that the writing system functions in a cascading manner (Delattre, Bonin, & Barry, 2006; Kandel et al., 2013; Olive, Alves, & Castro, 2009; Olive & Kellogg, 2002; Roux, McKeff, Grosjacques, Afonso, & Kandel, 2013). A writer can synchronously be engaged in transcribing a word and processing the next word. Therefore, interferences between different levels of handwriting are likely to occur during movement execution (Fayol & Lété, 2012). BODY.INTRODUCTION.AUTOMATICITY OF HANDWRITING, WORKING MEMORY, AND TEXT QUALITY: Low level components of handwriting implicate an integration and coordination of spelling knowledge, allographic representations and the execution of fine-motor movements (Berninger, 2009; Berninger & Swanson, 1994). For young children at the very beginning of their handwriting experience, each of these tasks is attention demanding and working memory resource consuming (Graham, Berninger, Abbott, Abbott, & Whitaker, 1997). The more this integration is automated, the lower the amount of the cognitive load is generally expected to be necessary (Bourdin & Fayol, 1994; Fayol & Lété, 2012) since handwriting automation is assumed to save resources for the remaining processes involved in text production (Grabowski, 2010; Graham et al., 1997; McCutchen, 1996, 2011). Children whose handwriting is not yet automated are easily overwhelmed with the more complex tasks of text production because of the demands on cognitive resources imposed on them, resulting in poor text production outcomes (e.g. Berninger & Swanson, 1994; Christensen & Jones, 1999; Graham, 1990). Accordingly, a training of handwriting over 8 weeks evidenced improvements both of the quality and quantity of written texts among adolescents suffering from poor orthographic-motor integration while the matched control group (whose students were instructed to write a diary) did not improve noticeably (Christensen, 2005). As Kim and Schatschneider (2016) demonstrated that working memory is a strong predictor of text quality already among first graders, handwriting training seems to be a promising method to improve the text quality early in the school career. Over the last years, this assumption could be confirmed for children with and without disabilities: automaticity trainings of transcription skills as handwriting lead to improvements in both text length and quality (Graham, McKeown, Kiuhara, & Harris, 2012). In their intervention study among Portuguese second graders, Alves et al. (2015) showed that a handwriting intervention group reached not only better handwriting fluency and text quality, but performed longer bursts with shorter pauses while writing texts when compared to keyboarding students. The handwriting training lasted over 10 weeks and included a pure functional graphomotor "warm up", followed by more and more challenging exercises, i.e. using the trained letters within words, sentences and stories. The comparison groups received similar trainings in spelling or keyboarding. BODY.INTRODUCTION.WORKING MEMORY, AUTOMATICITY OF HANDWRITING AND SPELLING: There is also some evidence that cognitive load is not only devoted to the higher-level processes of writing, but also to transcription such as spelling. Spelling requires the retrieval of orthographic knowledge about the correct letters and their sequences in spoken or written words involving both orthographic long-term memory and working memory resources (Buchwald & Rapp, 2009). According to Tainturier and Rapp (2001), two sets of processes are involved in spelling that do not function independently from one another: among frequent words the letter sequences are directly retrieved from the so-called orthographic lexicon (lexical route), whereas for rare or novel words, the letter sequences are derived by a phonology to orthography conversion system (sublexical route). From this point of view, we assume that spelling capability is affected by the phonological component of working memory. However, it has been demonstrated that the visual-spatial modality of working memory has an additional effect on children's spelling (Bourke, Davies, Sumner, & Green, 2014; Fischbach, Könen, Rietz, & Hasselhorn, 2014). Sumner, Connelly, and Barnett (2013, 2014) have investigated the impact of spelling capabilities on handwriting production in a copy task by comparing children with and without dyslexia. They found many more within-word pauses (and constrained productivity and fluency for that reason) among the dyslectic children compared to the spelling-ability matched children. Interestingly, children with dyslexia were able to write fast, but paused longer than the controls within-words, probably due to cognitive demands required by spelling information processing and poor lexical representations. It can therefore be concluded that handwriting fluency, as well as working memory, are both positively related to spelling capacity. BODY.INTRODUCTION.HANDWRITING AND VISUAL-MOTOR INTEGRATION: Visual-motor integration (VMI) skills proved to be associated with legibility (e.g. Tseng & Murray, 1994; Volman, van Schendel, & Jongmans, 2006), however, research using the Developmental Test of VMI did not find an association with handwriting difficulties in young children (Marr & Cermak, 2002) or with handwriting dysfunction in older children (Goyen & Duff, 2005). Nevertheless, there is some evidence that VMI is related to academic performance in reading and writing (Kulp, 1999; Sortor & Kulp, 2003). Therefore, we included this variable to control for its impact on text quality. BODY.INTRODUCTION.GENDER EFFECTS: Handwriting fluency, automaticity and working memory are not the only factors influencing the quality of texts produced by children. Research has revealed that girls produce better and longer texts than boys (e.g. Kim, Al Otaiba, Wanzek, & Gatlin, 2015), therefore, the gender gap is an additional factor that has to be considered when predicting text quality. Berninger and Fuller (1992) investigated first, second and third graders and found that girls produced more words and clauses in narrative and expository composition than boys. Therefore, gender differences in transcription skills could at least partly explain the text quality differences. In addition, regarding the fact that girls have more positive attitudes than boys towards writing (Knudson, 1995; Lee, 2013), such motivational factors have explained additional variance in text quality in some studies (e.g. Knudson, 1995) but not in others (e.g. Graham, Berninger, & Fan, 2007). BODY.INTRODUCTION.TRAINING OF TRANSCRIPTION SKILLS: Because of its important role as a transcription skill, handwriting has to be trained not only to become as legible as possible, but also to become more and more fluent. Fluency of handwriting means that a person is able to write in an automated way so that he or she does not have to think about how to form a letter or how to join movements of frequently used syllables. This results in an economic and fast way of personal handwriting. Automated movements show a particular shape of a smooth and regular course of velocity control: the ideal handwriter needs only one increase and decrease of speed per stroke unit (Mai & Marquardt, 1999). As shown by Graham, Berninger, Weintraub and Schafer (1998) and by Wicki, Hurschler Lichtsteiner, Saxer Geiger and Müller (2014), the development of both legibility and fluency of handwriting is not yet finished at the end of the second grade. There is more training needed over the following years of schooling. In their meta-analysis, Troia and Graham (2009) published 17 recommendations about the most efficient ways of teaching handwriting, e.g. that in the primary grades, 75–100 min per week should be used for handwriting instruction, that letters sharing common strokes (e.g., a, d, and g) should be grouped together, and that the children should be monitored and immediately get help to form letters better when they are illegible. Teachers are requested to allow children to develop their own handwriting style and to ask them to self-evaluate their handwriting and to improve it continuously. While traditional handwriting lessons used to focus on legibility of handwriting, the importance and the knowhow of teaching handwriting fluency is not yet well established. Santangelo and Graham (2016) recently found that in general, handwriting training is more efficient than no treatment. Individualizing instruction and using digital technology improved legibility, but no effects were found for fluency. A study by Berninger and co-authors (Berninger et al., 2006) on different ways to teach letters is important to understand why it is not useful to let children just copy letters directly from a model: In order to gain an automatized motor program, children need to trace a letter by visual arrows, getting verbal mediation, but afterwards, they are supposed to reproduce it from memory. Troia and Graham (2009) recommend further trainings in fluency by frequent writing and speed trials. Isolated motor trainings are inferior to handwriting training (Santangelo & Graham, 2016), accordingly, insights from promoting motor learning, e.g. the model of differential motor learning by Frank, Michelbrink, Beckmann and Schöllhorn (2008) or the principles of Neuromotor Task Training (Schoemaker & Smits-Engelsman, 2005) have been included in handwriting education (Jurt Betschart & Hurschler Lichtsteiner, 2017) and therapy. However, it has not yet been examined if they are really enhancing handwriting fluency among children with motor disabilities. A meta-analysis by Hoy, Egan, and Feder, (2011) proved that instructions of at least 20 lessons were effective, but the interventions focused on treatments by occupational therapist for children with handwriting difficulties. So far, there are only a few German studies investigating children's handwriting (e.g. Grabowski, Weinzierl, & Schmitt, 2010; Mahrhofer, 2004; Nottbusch, 2008; Sattler & Marquardt, 2010; Speck-Hamdan, Falmann, Hess, Odersky, & Rüb, 2016) and almost none looking at the link between transcriptions skills and text quality. This is unsatisfying because on the one hand German language has a more complex syllabic structure than Romance languages, as depicted by Seymour, Aro, and Erskine (2003), but on the other hand the orthographic depth of the German language is shallower than that of English for instance, so findings of previous studies in other languages may not be directly transferrable. In regard to written language acquisition in German-speaking countries, there are different ways of learning how to read and write in general and how to write by hand in particular. In the German-speaking part of Switzerland, a new handwriting style has been taught since 2001 in some schools locate. After positive research results confirming that children with the new handwriting type were able to write more legibly and fluently and showed more motivation to write than with the former one (Wicki & Hurschler Lichtsteiner, 2014), the so-called "Basisschrift" was established in almost every canton. This handwriting style has changed teaching methods, because the children now start with a beginners' alphabet of printed letters, then learn how to connect the most common letters fluently by garlands (joins on the baseline). Finally, students get support to develop their individual handwriting style (Jurt Betschart & Hurschler Lichtsteiner, 2017). In the German part of Switzerland, no studies about handwriting instruction nor any implications on language acquisition and text quality in particular have been published so far. BODY.INTRODUCTION.HYPOTHESES: With respect to the different trainings carried out over 5 weeks, we expected that the combined handwriting/spelling training would improve the fluency of handwriting (speed and automaticity) and advance correct spelling as well as the text quality (i.e. number of ideas, text organization) to a higher degree than the handwriting only or a spelling only training because of the broader approach of the combined training, which included two transcriptions skills.In addition, we expected that the handwriting only and the spelling only training groups would both outperform the reading control group regarding handwriting fluency, spelling and text quality. We also expected that the aforementioned group differences would sustain over a period of 3 months after termination of the intervention.Irrespective of the trainings, we expected that text quality would be independently affected by handwriting fluency and automaticity, spelling, working memory, and gender. Text quality is assumed to be higher among children, indicating higher handwriting fluency and automaticity, improved spelling and working memory capabilities. In addition, text quality is assumed to be higher among girls than among boys. BODY.METHOD.DESIGN: A randomized controlled field study with pre-test, post-test and follow-up-test measurements was conducted. The sample included one main intervention group (a combined handwriting and spelling training), two comparison groups (handwriting training and spelling training) and a control group (reading fluency). The participants were typically developing third-graders (N = 175). This sample size was higher than the required sample size (N = 158) calculated by means of the GPower software (Faul, Erdfelder, Lang, & Buchner, 2007) assuming an effect size f(V) = 0.26 and alpha error = 0.05 (given 4 groups and 3 measurements, and repeated measures ANOVA). In order to control for cluster effects, each class was randomly divided in two parts, whereby the minority of every class was allocated to the main intervention and the majority randomly to one of the comparison groups or to the control group (Fig. 1). Most measures were assessed three times, however, working memory capacity and visual-motor integration were assessed only during the pre-test.Fig. 1Assignment of children to the groups, N = 175 BODY.METHOD.PARTICIPANTS: Principals from elementary schools in the Canton of Lucerne in the German speaking part of Switzerland were asked to help the research team in recruiting the participating classes. The participating teachers had to give their consent of attending to four preparatory meetings and organizing the testing and training activities. They also had to find a second teacher for the training phase who was usually the associated teacher for special needs education. All children in the classes were included in the curriculum-based trainings. 202 children and their parents were asked to give their active consent for the additional tests and the use of the data; in 21 cases, the consent was refused. Because the average number of pupils in each class was lower than expected, it took 11 instead of 9 classes to reach the necessary sample size. 6 of the 181 participants moved away during the intervention time, which resulted in 175 children who attended the whole training program and all of the tests (attrition = 3.3%). Within the participants, there were no exclusions due to learning disabilities or other issues, but in order to control for special effects, all possible variables were noticed. 22 children (12.6%) attended integrative special needs education; 11 children (6.3%) received therapies such as psychomotor therapy. These cases were evenly distributed over the four intervention groups. 20 children (11.4%) attended L2 courses in German with uneven distribution among the groups: 17 of them were in the combined handwriting and spelling or in the handwriting group; due to the low numbers, a statistical test of this difference was not possible. Regarding age (Mage = 9.0 years, SD = 0.4 years, age range = 7.5–10.2 years), the children of the four intervention groups did not differ significantly. Demographic characteristics such as gender, handedness and language spoken were equally distributed over the four groups (see Table 1).Table 1Demographic characteristics of the sampleVariableCombinedHandwritingSpellingReadingN%n%n%n%n%χ2p17578343627Gender0.599n.s. Female8950.93848.71852.92055.61348.1 Male8649.14051.31647.11644.41451.9Handedness6.076n.s Right handers15789.77494.92882.43083.32592.5 Left handers1810.345.1617.6616.727.4Languages4.879n.s. Swiss German speaking only12973.76178.22161.82980.61866.7 Two or more languages4626.31721.81338.2719.4933.3 BODY.METHOD.MATERIALS (DIGITIZER): Handwriting measures were recorded and analyzed by means of the software package CSWin (Mai & Marquardt, 2007) on a personal computer (Lenovo ThinkPad T61p Type 6457-7WG), which was connected to a digitizing tablet (Wacom Intuos 4 Pen Tablet PTK-640 and Intuos Inking Pen). The sampling frequency was 200 Hz and the accuracy was 0.1 mm in both x and y directions. Position data were recorded even if the stylus was lifted (less than 10.0 mm) above the tablet due to the inductive method of measurement. Nonparametric regression methods (kernel estimation) are part of the mathematical procedures of the program (Marquardt & Mai, 1994) in order to calculate and to smooth velocity and acceleration signals. BODY.METHOD.INTERVENTION: 7 months prior to intervention, four meetings with the participating teachers took place: All participants had to plan the same quantity and quality of instruction and training regarding writing, German orthography and handwriting training throughout the curriculum. In addition, every teacher got an introduction into the didactic structure of the handwriting lessons as well as the materials necessary to prepare standardized lessons in the time period from summer 2015 to the end of January 2016 when the intervention started. The participating teachers (n = 11) and their students were randomly assigned to the different intervention and control conditions, which lasted 20 units (15 min per unit) distributed over 5 weeks (4 units/week). The respective trainings were highly standardized and held during ordinary school hours by the participating teachers who had been instructed in advance. The teachers had to check for an implementation as full as possible. Within the materials, the last page (for the last day) included a feedback sheet for the participating pupils in order to assess their motivation. We know that effective handwriting training should always be followed by meaningful tasks like writing or at least exercises including language actions (playing with words, little learning games like riddles) (Alves et al., 2015; Berninger & Richards, 2002). However, trainings for handwriting skills often include (sometimes unintended) orthographic challenges and spelling training can be very demanding for some children when it takes a lot of handwriting. This is why we tried to separate these training conditions from the beginning. Even the combined training offered a simple start without cognitive overload followed by different transfer opportunities. BODY.METHOD.INTERVENTION.COMBINED HANDWRITING AND SPELLING TRAINING: Completely new training materials were developed for the combined intervention and for the handwriting intervention as well. Due to the semi-cursive handwriting type used in the German-speaking part of Switzerland, the material did not focus on the repetition of the alphabet (since the production of individual letters is presumed to be already automatized at that age), but on the fluent production of the connections between the letters, specifically on the connections as realized by garlands (joins on the baseline). According to Berninger and Richards (2002), the training was planned to be taught explicitly in a regular manner, by providing short and daily trainings of 15 min followed by writing activities. The daily intervention program was presented by a two-sided sheet with two A4-sized pages of material on each side starting with a finger-warm-up exercise (scribbling strokes and circles) followed by a graphomotor training of a selected pair of letters to be joined fluently under different conditions regarding speed, pressure, size and visual control according to Frank, Michelbrink, Beckmann and Schöllhorn (2008). Every week, a group of similar patterns (e.g. day 1 e-n, day 2 e-m, day 3 e-r) was offered. The last day was dedicated to repetitions of this group. These trainings had to be completed in broad colorful bands instead of rulings. In the combined handwriting and spelling condition, the task in the second part was to put these patterns of 2–3 joined letters into use within a spelling task, which was a training mostly focusing on morphemes, or to use these patterns directly by writing words or sentences. To make sure that the children spent enough time on the language-based part the last 5 min had to be strictly spent on the pages of the second part. BODY.METHOD.INTERVENTION.HANDWRITING ONLY TRAINING GROUP: In the handwriting group, the tasks consisted of a graphomotor training (the same as page 1 and 2 of the combined group supplied with additional tasks of that type) without any combination with spelling training. BODY.METHOD.INTERVENTION.SPELLING ONLY TRAINING GROUP: The spelling training was based on a training developed by Leemann (2015). We applied the chapter focusing on morphemic structures (Leemann, 2015, pp. 19–44), which had been described as the third of three important principles of German orthography (Eisenberg & Feilke, 2001) and is part of the regular curriculum at that age. Exercises consisted of recognizing morphemic elements, analyzing words and constructing them by using morphemes. In order to avoid handwriting within these tasks, the children had to complete them without handwriting, e.g. by marking morphemes using text markers or connecting questions and solutions by cues. BODY.METHOD.INTERVENTION.READING TRAINING CONTROL GROUP: The training for reading fluency consisted of two parts (reading skills and reading fluency training) of the training developed by Kruse, Rickli, Riss, and Sommer (2014). Handwriting activities were omitted during these trainings. The intervention protocols indicated a mean number of training units of 19.3 (SD = 1.4) for the total sample. The mean number of training units varied from 19.0 to 19.8 over the four groups and did not differ significantly from each other. BODY.METHOD.PROCEDURE: Data collection of the pre-test took place at the end of January 2016. The intervention started midst February and lasted 5 weeks. During the subsequent week, the post-tests were carried out. After a break of 11 weeks, the data collection of the follow-up-test took place midst June 2016. Most tests were group-administered by the teachers (spelling test, copy task, writing a 30'-text, visual motor integration), whereas two tests (Working Memory Test AGTB-5, Hasselhorn et al., 2012; and CSWin, Mai & Marquardt, 2007) were executed as single tests by the data collection team consisting of two researchers and five scientific assistants. Legibility of the handwriting was assessed by analyzing the copied texts by a rating system; however, since this variable is not focused on in this paper, we do not describe this measure in detail in this study. BODY.METHOD.TASKS AND MEASURES.PERSONAL DETAILS OF PARTICIPANTS: Details on children's handedness, gender, age, languages spoken were collected by the participating teachers and submitted to the research team. BODY.METHOD.TASKS AND MEASURES.HANDWRITING FLUENCY MEASURED ON THE DIGITIZER: The handwriting tasks were administered on a digitizing tablet and included a range of 17 items starting with basic handwriting movements, letter, and combinations of two joined letters, syllables, morphemes, non-sense words and common words. The children also had to copy sentences, carry out a small dictation and write two separate 3-min short texts. The first was about a fantasy theme ("If I could conjure", "If I were rich", "If I were a super star"), the second was a prescription ("This is my way to school", "My favorite game", "My favorite pet"). All tablet items were written with a regular inking pen on a blank sheet of paper placed on the digitizing tablet. Within each task, we derived the number of inversions in velocity (NIV) and the stroke frequency (FREQ) from the digitizer. The number of inversions in velocity (NIV) indicates the average number of velocity changes occurring within writing strokes. Fluent handwriting requires only one velocity change per stroke (acceleration followed by deceleration) and therefore results in a NIV score that is approximately or exactly equal to 1, whereby 1 indicates perfect automaticity (Mai & Marquardt, 1999). When copying a sentence, the mean number of inversions in velocity of 4th graders amounted to 2.7 (SD = 2.1) in an earlier study (Hurschler, Saxer, & Wicki, 2010). Frequency (FREQ) refers to the number of upward and downward movements in 1 s calculated by CSWin (Mai & Marquardt, 1999). This measure seems more appropriate than assessing speed as stroke length per second (mm/s) (the latter strongly depends on a person's writing size). In our earlier study, the mean stroke frequency of 4th graders was f = 2.1 (SD = .6) when copying a sentence (Hurschler et al., 2010). BODY.METHOD.TASKS AND MEASURES.HANDWRITING MEASURES IRRESPECTIVE OF TABLET TASKS: The children had to execute a 5 min copying task where they were asked to write as fast and as legible as possible. Speed of handwriting was measured as the number of letters written within that task. The respective data of one class had to be excluded from the analysis because of violating the time limit. BODY.METHOD.TASKS AND MEASURES.VISUAL MOTOR INTEGRATION VMI (BEERY & BEERY, : The Developmental Test of Visual-Motor Integration (VMI) "is designed to assess the extent to which individuals can integrate their visual and motor abilities" (Beery & Beery, 2006, p.14). It consists of 30 graphic forms of increasing complexity that are to be copied successively. The scoring procedure follows the instructions as specified in the test manual. BODY.METHOD.TASKS AND MEASURES.WORKING MEMORY: At pre-test (t1), we administered three working memory sub-tests from the AGTB 5-12 (Hasselhorn et al., 2012). In addition to the two visual-spatial tasks (Corsi-Block and Matrix), a third task referring to the phonological loop (word-list recall-test, monosyllabic words) was administered. BODY.METHOD.TASKS AND MEASURES.SPELLING: In order to get three parallel test versions for spelling, we combined the age-appropriate items of the "Salzburger Rechtschreibetest: SLRT-II" (Moll & Landerl, 2014) with some age-appropriate items from the "Diagnostischer Rechtschreibtest DRT 3" (Müller, 2003). The resulting test versions were pilot tested in advance revealing comparable test difficulties. BODY.METHOD.TASKS AND MEASURES.TEXT QUALITY: The children were asked to write short experimental narrative texts according to a prompt (adapted from Kim et al. 2015). The three prompts linked to the pre-, post- and follow-up test were familiar to their everyday life ("This happened when I got home from school...", "This happened in our break...", "This happened when I was a child...") and were presented in a randomized order for each class. Children were allowed to write a brief mind-map of their ideas before writing. They were told that this time, their drafts would be the piece that would be evaluated and that there would be no phases of revision and/or clean copy. Each time, the teacher told the children the principal criteria of the later assessment in advance. The four criteria (1. My story has a gripping beginning, a middle part and a clear ending; 2. It is coherent without disruptive leaps; 3. It is told in a vivid and exciting way; 4. I use appropriate matching words and sentences) were derived from the curricular teaching materials. There was no help allowed by the teacher or by peers during production. After 30 min, all products—drafts and pieces of planning work—were collected and sent to the research team. The text quality was assessed by a rating procedure based on two German studies, Ko-Text (Kruse, Reichardt, Herrmann, Heinzel, & Lipowsky, 2012) and RESTLESS (Wild et al., 2016), meeting our requirements with respect to grade level and text type (narrative texts). The original rating consisted of 10 Likert-type items complemented by anchor examples. After the instruction of 6 raters and preliminary interrater tests, the applied rating system consisted of 7 items (coherence of topic, logic of action, cohesion, implicitness of the text, appropriate language, orientation towards basic patterns of narrative texts, and language risk). These items are outlined in detail in the Appendix. The text quality measure was calculated by two steps. First we combined the three coherence items (coherence of topic, logic of action, cohesion) into one coherence measure. Subsequently we averaged coherence and the remaining four items. In order to assess the interrater reliability, 19 texts (of the pre-test) were rated by all raters. Based on the average ratings, we calculated the intraclass correlation (ICC) average score by applying the two-way model. The resulting ICC of 0.97 indicates a good reliability. BODY.METHOD.TASKS AND MEASURES.MOTIVATION AND SELF-EVALUATION: Integrated in the last training sheet, the children were asked to complete a short survey of self-evaluation relating to level of program difficulty and their motivation to participate and were given the possibility to write an open formatted feedback. BODY.METHOD.DATA ANALYSIS: The hypotheses with respect to the intervention effects were examined as group × time interactions within the longitudinal data set (repeated measurements). We calculated them by means of repeated measures ANOVAs. Some departures from sphericity were identified by the Mauchly's test in SPSS. Since all violations were weak (epsilon > 0.75), we applied the Huynh–Feldt correction. As the handwriting measures derived from the digitizer (number of inversions in velocity and stroke frequency) were skewed and not normally distributed (as confirmed by Shapiro–Wilk tests), we calculated the logarithmic (base 10) values of these measures to meet the requirements for parametric statistics outlined before. BODY.RESULTS: In this section, we will first describe the level of fluency and automaticity of handwriting being revealed in the different tasks used in this study. Secondly, we will report the results concerning the intervention effects, and thirdly we will present our results with respect to the hypotheses that are irrespective of the intervention. BODY.RESULTS.FLUENCY OF HANDWRITING AMONG THIRD GRADERS: As the original values (before logarithmic transformation) are easier to understand (what these figures really mean), we will depict the means and standard deviations of the untransformed values in Table 2.Table 2Fluency of handwriting across timeMeasuret1t2t3M(SD)M(SD)M(SD)NIV 11.54(1.00)1.24(0.47)1.23(0.57)FREQ 13.34(1.17)3.69(0.95)4.02(1.00)NIV 22.74(3.17)2.04(2.21)1.59(0.88)FREQ 21.80(0.74)2.06(0.75)2.26(0.85)NIV 31.52(1.28)1.49(2.98)1.10(0.35)FREQ 33.01(1.14)3.40(1.00)3.62(0.91)NIV 41.26(0.55)1.35(0.69)1.35(0.69)FREQ 42.91(1.06)2.71(1.07)2.61(1.05)NIV 51.13(0.37)1.13(0.40)1.07(0.17)FREQ 53.71(1.20)3.71(1.20)3.63(1.03)NIV 61.25(0.65)1.18(0.44)1.23(0.51)FREQ 62.99(0.76)3.08(0.72)3.05(0.77)NIV 71.09(0.18)1.06(0.12)1.04(0.09)FREQ 73.63(0.71)3.85(0.70)3.93(0.62)NIV 82.41(0.18)2.32(1.46)2.12(1.31)FREQ 81.88(0.62)1.82(0.55)2.02(0.57)NIV 93.25(2.25)2.17(1.27)2.37(1.48)FREQ 91.54(0.53)1.81(0.51)1.86(0.56)NIV 102.56(1.48)2.05(1.26)1.83(0.92)FREQ 101.82(0.57)1.99(0.58)2.22(0.66)NIV 112.23(1.27)2.10(1.11)1.83(1.00)FREQ 112.08(0.64)2.12(0.67)2.46(0.75)NIV 121.78(1.02)1.73(1.03)1.55(0.82)FREQ 122.51(0.80)2.45(0.82)2.80(0.88)NIV 131.69(0.71)1.48(0.53)1.37(0.45)FREQ 132.49(0.62)2.65(0.59)2.91(0.58)NIV 141.58(0.64)1.48(0.51)1.38(0.37)FREQ 142.65(0.68)2.70(0.60)3.01(0.58)NIV 151.63(0.66)1.52(0.58)1.38(0.43)FREQ 152.62(0.66)2.70(0.67)3.03(0.61)NIV 161.24(0.25)1.22(0.25)1.18(0.16)FREQ 163.52(0.67)3.59(0.67)3.91(0.62)NIV 171.44(0.56)1.40(0.41)1.29(0.30)FREQ 172.92(0.64)2.94(0.63)3.24(0.56)N = 174 − 175NIV = Number of Inversions in Velocity; FREQ = Stroke Frequency; 1 = scribbling; 2 = finger movements; 3 = finger movements-faster; 4 = wrist movements; 5 = wrist movements (faster); 6 = combined finger and wrist movements; 7 = combined finger and writ movements (faster); 8 = garlands; 9 = double loops; 10 = syllables; 11 = nonsense words; 12 meaningful words; 13 = 3′ composition (based on a preliminary version); 14 = 3′ composition (descriptive text-without a draft); 15 = sentence writing (normal pace); 16 = sentence writing (as fast as possible); 17 = sentence dictation According to Marquardt (2011), a handwriting movement is almost automated if the number of inversions in velocity is lower than 1.5. Regarding the data at the pre-test, it is remarkable that for some tasks, the children did already show automatized movements. As expected, they very well managed to perform the simplest basic movements as wrist movements (NIV 4, NIV 5) and combined finger and wrist movements (NIV 6, NIV 7). This is not the case in regard to patterns like garlands (NIV 8) and double loops (NIV 9) or while writing syllables (NIV 10), words (NIV 11, NIV 12) and composing short texts (NIV 13, NIV 14). However, when asked to write a sentence as fast as possible (NIV 16) or to write a difficult sentence by dictation (NIV 17), they achieved very low values indicating high automaticity. Over time, almost all movements were at an automatized level at t2, even the short texts, except at the first trial of finger movements (NIV 2) and the garlands, double loops, syllables and words. In general, a clear linear improvement over time is discernible by visual inspection only. The stroke frequencies mirror the number of inversions in velocity findings described above: They are in general already high from the beginning of the study at pre-test and further improve from t1 to t3. BODY.RESULTS.INTERVENTION EFFECTS: In order to avoid a multiple testing problem (accumulation of alpha error), we restricted the testing of the expected intervention effects on fluency to the movements (tasks) that were not yet fully automated at t1, i.e. double loops, a syllable (i.e. "neu"), words (i.e. "akir" and "Falle") and the two composed texts. Thus, twelve repeated measures ANOVAs were separately carried out using the fluency measures (automaticity and frequency) of six of the mentioned tasks as dependent variables. We found reliable time effects in almost all analyses but-with two exceptions—no intervention effects. The three intervention groups, as well as the control group, wrote more fluently (i.e. with improved automaticity, higher stroke frequency) over time. For example, the automaticity of handwriting (measured by the NIV score) when composing a text improved significantly over time (F(1.8,306.9) = 26.9, p < 0.001, ηp2 = 0.14), however, in general, there was no group*time interaction, i.e. the improvements of the combined intervention group did neither outperform the improvements of the spelling only nor the handwriting only training group nor did they differ from the improvements of the reading control group. One of the exceptions mentioned above was a marked increase of fluency in the double loops tasks observed in the combined and in the pure handwriting group from pre-test to post-test, which outperformed the spelling training and the reading groups as indicated by the significant interaction term intervention*time (F(3,171) = 2.7, p < 0.05, ηp2 = 0.05). As depicted in Fig. 2, the differences did not persist completely until the follow up, resulting in a nonsignificant interaction term when the third measurement was also included in the model.Fig. 2Course of automaticity (double loops) among interventions groups The second exception concerned stroke frequency when completing the task referring to writing the German word "Falle". Here, we found an intervention × time interaction (F(6,342) = 3.8, p = 0.001, ηp2 = 0.06) besides an intervention effect (F(3,171) = 3.2, p < 0.05, ηp2 = 0.05) and a time effect (F(2,342) = 12.3, p < 0.001, ηp2 = 0.07). Interestingly enough but completely unexpected, the respective means indicated that the stroke frequency declined among the combined and the handwriting training group from t1 to t2 and recovered from t2 to t3, i.e. showing a paradox intervention effect, while the spelling group and the reading group did improve their stroke frequency quite linearly over time. With respect to our hypothesis that assumed intervention effects on spelling and text quality, we calculated two additional repeated measures ANOVAs including this time spelling and text quality respectively as dependent measures. Again, these analyses failed to discern any group differences or intervention effects. However, we found a time effect showing that text quality had improved over time (F(2,338) = 6.1, p < 0.01, ηp2 = 0.04). BODY.RESULTS.PREDICTING TEXT QUALITY IRRESPECTIVE OF THE INTERVENTION: Our third hypothesis relates to the prediction of test quality regardless of group membership. We calculated a hierarchic regression analysis on text quality (measured at t3) by entering gender as the first predictor. In the second step, we entered two working memory measures (t1) and visuo-motor integration (t1) and in the final step, we entered the transcription measures, i.e. number of inversions in velocity (t3) and speed (t3) as well as spelling (t3). In order to investigate the influence of a clear-cut low automaticity, the number of inversions in velocity measure used for that analysis was dichotomized above one standard deviation (see Table 1). The final regression equation was significant indicating that the included variables are able to predict text quality (adj r2 = 0.21). The results presented in Table 3 reveals that gender, the phonological part of the working memory, spelling and the dichotomized NIV contributed to this prediction while the visual part of the working memory, visuo-motor integration and writing speed did not.Table 3Summary of hierarchical regression analysis for variables predicting text qualityVariableModel 1Model 2Model 3 B SE B β B SE B β B SE B β Gender− 0.270.07− 0.28**− 0.290.07− 0.31**− 0.230.07− 0.25**WM word span0.200.050.26**0.150.050.20**WM visuo-spatial sketch pad− 0.000.020.020.000.030.02VMI0.020.010.080.020.010.09Handwriting speed0.000.000.02NIV Composing− 0.350.15− 0.17*Spelling− 0.020.00− 0.23** R 2 0.080.160.20F for change in R214.90**5.35**6.03**NIV composing is a dichotomized variableWM working memory; VMI Visuo-motor integration*p < 0.05. **p < 0.01 BODY.DISCUSSION.INTERVENTION EFFECTS: Third graders' handwriting performance is interesting to analyze as they are in the midst of the learning process: They are more advanced than real beginners because they have mastered the first step of using the corresponding link from phonemes to graphemes and they know how to form the letters, but are not yet fully automatized since they are not able to write words and sentences without paying attention as skilled writers do. Therefore, this intervention study was designed to investigate the impact of a training program that aims to improve the transcription skills of third graders. Improved fluency is assumed to have a positive impact on text quality as previous studies have demonstrated. In line with our assumptions, we could only demonstrate a short-term intervention effect on fluency with respect to the automaticity improvements of double loops that, however, did not persist until t3. Besides that, the intervention did not improve fluency among the two handwriting groups to greater extent than among the remaining groups (spelling and reading). In contrast, we found a paradox effect indicating that the handwriting training could also have contributed to a reduction of fluency as evidenced by the results related to the task of writing a meaningful word. It is likely that the handwriting training temporarily promoted more deliberate movements among some children and not more automaticity in the first place. Another explanation for the missing intervention effect is the fact that the children's automaticity e.g. when writing sentences was already very well developed at the beginning of the study. Irrespective of whether they really realized the promoted garlands as a principle to increase handwriting fluency in their own handwriting (which would be an interesting qualitative further investigation), after reaching a certain automaticity in their own personal style, to learn and integrate new patterns could interfere with further improvements of fluency in the short-term. Under this premise, further improvements could not so easily be achieved by means of a training lasting 5 weeks or 20 units. Furthermore, it is quite difficult to prevent a control group like our reading group from handwriting "training" over 5 weeks of schooling since handwriting is part of everyday activities beyond the intervention units. However, it is possible that a program lasting longer than 5 weeks and more training units can enhance transcription skills among third graders as was the case in the study conducted by Alves et al. (2015). As the intervention did not yield differential effects on fluency, it is theoretically congruent that we also did not find any intervention effects on text quality since the former was thought to be a precondition of the latter. The fact that we did not find an effect on the spelling training is perhaps less surprising. The improvements in spelling develop slowly and are hardly measurable within several weeks. BODY.DISCUSSION.AUTOMATICITY OF HANDWRITING, WORKING MEMORY, SPELLING, AND TEXT QUALITY: As outlined above transcription skills facilitate composing especially among young children because at that age handwriting is not yet automated and therefore working memory resource consuming (Graham, Berninger, Abbott, Abbott, & Whitaker, 1997). In line with this assumption the multiple regression analysis revealed that text quality positively was both related to the phonological loop component of working memory and to transcription measures such as fluency and spelling. In some contradiction with previous research, however, text quality was independent from the visuo-spatial component of working memory. With respect to transcription, interestingly, speed writing per se did not predict text quality whereas it was a dichotomized variable consisting of very low automaticity versus normal to good automaticity (measured as number of inversions of velocity) when writing a short text that emerged as an independent predictor of text quality. Thus, children whose cognitive resources are consumed by the transcription process because of very low automaticity and reduced spelling capacity lack that capacity when composing. This, in turn, is one possible reason for low text quality, a finding that is consistent with Prunty, Barnett, Wilmut and Plumb (2016), who investigated children with developmental coordination disorder, although they used other measures of handwriting fluency. BODY.DISCUSSION.GENDER: Gender successfully predicted text quality. Referring to gender it is well known that boys' reading fluency competencies are lower than girls' (Bos, Tarelli, Bremerich-Vos, & Schwippert, 2012), and also the text quality of boys seems to be constantly lower than that of girls (Kim et al., 2015). Thus, we replicated previous results and controlled for the gender effect as we entered gender first into the regression. BODY.CONCLUSION: According to Santangelo and Graham (2016), teaching handwriting is an important duty over all primary school years. Although they advocated an individualized instruction taking into account different stages of graphomotor development and the capabilities to deal with new patterns, our design followed an experimental logic by controlling as many factors as possible. Therefore, we have to admit that our trainings among third graders do not fulfil the demands of an individualized instruction. As a recommendation to the involved schools and to teachers of third graders, we therefore propose to start the teaching on how to join letters by garlands earlier (that means at the beginning of the third class) when children are not yet fully automatized in their handwriting, to observe learning progress carefully and to offer suitable individualized training as it is realized in the official materials in the meanwhile (Jurt Betschart, Hurschler Lichtsteiner, & Reber, 2016).

TITLE: Intrathecal Morphine Plus General Anesthesia in Cardiac Surgery: Effects on Pulmonary Function, Postoperative Analgesia, and Plasma Morphine Concentration ABSTRACT.OBJECTIVES:: To evaluate the effects of intrathecal morphine on pulmonary function, analgesia, and morphine plasma concentrations after cardiac surgery. ABSTRACT.INTRODUCTION:: Lung dysfunction increases morbidity and mortality after cardiac surgery. Regional analgesia may improve pulmonary outcomes by reducing pain, but the occurrence of this benefit remains controversial. ABSTRACT.METHODS:: Forty-two patients were randomized for general anesthesia (control group n=22) or 400 μg of intrathecal morphine followed by general anesthesia (morphine group n=20). Postoperative analgesia was accomplished with an intravenous, patient-controlled morphine pump. Blood gas measurements, forced vital capacity (FVC), forced expiratory volume (FEV), and FVC/FEV ratio were obtained preoperatively, as well as on the first and second postoperative days. Pain at rest, profound inspiration, amount of coughing, morphine solicitation, consumption, and plasma morphine concentration were evaluated for 36 hours postoperatively. Statistical analyses were performed using the repeated measures ANOVA or Mann-Whiney tests (*p<0.05). ABSTRACT.RESULTS:: Both groups experienced reduced FVC postoperatively (3.24 L to 1.38 L in control group; 2.72 L to 1.18 L in morphine FEV1 (p=0.085), group), with no significant decreases observed between groups. The two groups also exhibited similar results for FEV1/FVC (p=0.68) and PaO2/FiO2 ratio (p=0.08). The morphine group reported less pain intensity (evaluated using a visual numeric scale), especially when coughing (18 hours postoperatively: control group= 4.73 and morphine group= 1.80, p=0.001). Cumulative morphine consumption was reduced after 18 hours in the morphine group (control group= 20.14 and morphine group= 14.20 mg, p=0.037). The plasma morphine concentration was also reduced in the morphine group 24 hours after surgery (control group= 15.87 ng.mL−1 and morphine group= 4.08 ng.mL−1, p=0.029). ABSTRACT.CONCLUSIONS:: Intrathecal morphine administration did not significantly alter pulmonary function; however, it improved patient analgesia and reduced morphine consumption and morphine plasma concentration. BODY.INTRODUCTION: Lung dysfunction after cardiac surgery is the most frequently reported cause of postoperative morbidity.1,2 In fact, postoperative atelectasis occurs more frequently in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) than in any other type of surgery3 In patients undergoing cardiac surgery, death caused by respiratory complications has been reported as being more frequent than that due to cardiac causes.4 Adequate perioperative analgesia enables full expansion of the chest, thereby contributing to reduce lung collapse in spontaneously breathing patients. This therapeutic intervention may reduce morbidity, hospital costs, and length of stay, thus improving patient quality of life and reducing the incidence of chronic pain.5 The impact of postoperative regional analgesia on major outcomes in non-cardiac surgery has been studied.6–8 Small doses of opioids administered to the central nervous system provide adequate analgesia, reducing the risks of intravenous analgesic administration, such as respiratory depression, pruritus, nausea, and vomiting. However, in the cardiac surgical population, regional anesthesia techniques are not routinely applied due to the scarcity of supporting studies and administration risks associated with preoperative systemic anticoagulation9 Low doses of intrathecal morphine used to control postoperative pain has been shown to promote prolonged analgesia,5 and has been associated with a lower risk of hematoma formation compared to the epidural technique.10 Intrathecal morphine has been studied for several years in patients undergoing cardiac surgery, but these studies were primarily directed towards evaluation of pain control and hemodynamic stability, and did not consider its possible impact of intrathecal morphine on respiratory function recovery.11 The objective of this study was to evaluate the effects of intrathecal morphine on pulmonary function, postoperative analgesia, and morphine plasma concentrations in patients undergoing coronary artery bypass surgery (CABG) with cardiopulmonary bypass (CPB) in comparison to standard intravenous analgesia. BODY.MATERIALS AND METHODS: After approval of the hospital ethics committee and collection of free informed consent, forty-two patients undergoing CABG with CPB and ranging in age from 18 to 80 years were included. Exclusion criteria were an ejection fraction below 40%, contraindications to neuraxial blockage, coagulopathy, use of low-weight heparin, warfarin, intrathecal morphine, or a platelet aggregation inhibitor other than aspirin, systemic or local infection, combined procedures, and patients with a specific contraindication to the medication employed in this study. Preoperative aspirin use was not an exclusion criterion. Preoperative surgical risk was evaluated using Higgins Surgical Risk Scale for cardiac surgery, and patients were considered: (1) minimum risk, (2) low risk, (3) moderate, (4) high, (5) extreme risk12. The patients were randomly assigned to receive general anesthesia with prior administration of intrathecal morphine at a dosage of 400 μg (morphine group n=20) or general anesthesia alone (control group n=22) according to a simple computer-generated list. BODY.MATERIALS AND METHODS.ANESTHESIA AND SURGICAL PROCEDURES: On the day of the operation, the patients received 1 to 2 mg kg −1 of midazolam orally 30 minutes before surgery, up to a maximal dose of 15 mg. Patients were monitored by continuous ECG and ST-analysis, pulse oximetry, and an invasive arterial line inserted in the right radial artery. In the morphine group, a 400-μg intrathecal injection of morphine was administered using a 27-gauge spinal needle in the L3-L4 space prior to induction of general anesthesia. If intrathecal puncture was not successful after two attempts, the patient was excluded from the study. After pre-oxygenation, general anesthesia was induced using 0.3 mg/kg of hypnomidate, 0.1 mg kg−1 of pancuroniun bromide, and 0.5 μg kg−1 of sufentanil first as a bolus, followed by a continuous infusion at 0.5 μg kg−1 h−1. Mechanical ventilation was initiated using a Cícero ventilator (Dräger®, Germany) with a tidal volume of 8 ml kg−1, respiratory frequency of 12 ipm, oxygen inspired fraction of 0.6%, and 5 cm of H2O PEEP. A nasoesophageal thermometer, bladder catheter, and central venous catheter were inserted after anesthesia induction. When judged necessary by the attending anesthesiologist, a pulmonary artery catheter was placed in the right internal jugular vein. Immediately after intubation and after CPB weaning, a lung expansion maneuver using an airway pressure of 30 cmH2O for 20 seconds was performed to revert any intraoperative lung collapse. During CPB, hypnosis was maintained with a propofol infusion aimed at maintaining a calculated plasma propofol concentration of 2.5 ug ml−1 according to the Marsh model13. All patients received 1 g of methylprednisolone intravenously before initiation of CPB according to the institutional protocol. Sufentanil infusion was terminated at the moment of skin suture. After arrival in the intensive care unit (ICU), a patient-controlled analgesia pump (PCA) programmed for a 1 mg bolus of morphine with an administration interval locked at 5 minutes and free demand was installed. Dipyrone (30 mg kg−1) was administered if patients presented persistent pain despite the use of PCA. Tracheal extubation was performed when patients were fully awake and responsive to verbal commands, as well as when the peripheral oximetry was greater than 94%, spontaneous respiratory frequency was greater than 10, temperature was greater than 36° C, and the patient was hemodynamically stable such that they were bleeding less than 100 ml/h. BODY.MATERIALS AND METHODS.VENTILATORY AND PAIN SCORE EVALUATION: Spirometry using an Easy One® portable spirometer (Niche Medical, West Leederville, Australia) was also performed preoperatively in the first and second postoperative days (POD) to measure forced vital capacity (FVC), forced expiratory volume in the first second (FEV1), and thus the FEV1/FVC ratio. Arterial blood samples were collected during the preoperative period, immediately before surgery, after anesthesia induction, at the end of surgery, and in the first and second postoperative days to evaluate the PaO2/FiO2 ratio. Pain was evaluated using a visual numeric scale from zero to ten at rest, at the time of profound inspiration, and after cough at 3, 6, 12, 18, 24, and 36 hours postoperatively. Morphine cumulative consumption and solicitation was also evaluated at the same times listed above. All postoperative pain, respiratory function, and morphine utilization measurements were obtained by individuals who were not involved in patient randomization and anesthesia administration. Blood samples assessed for plasma morphine concentrations were collected intraoperatively, immediately after stopping sufentanil infusion, after 5, 15, 30, and 60 minutes, and after 3, 6, 12, 18, 24, and 36 hours in the ICU. The blood samples were centrifuged at 10.000 rpm for 10 minutes, and the supernatant was frozen at − 70 oC for later analysis. Plasma morphine concentrations were measured using a mass spectrophotometer. BODY.MATERIALS AND METHODS.SAMPLE SIZE AND STATISTICAL ANALYSIS: A sample size of at least 36 patients was deemed necessary to detect a statistically significant decrease (p < 0.05) in cumulative morphine consumption of 20% between the morphine and control group, assuming a standard deviation of 10 mg in a single-tail paired Student's t-test using G Power 3 software (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany). Statistical analyses were performed using SPSS 16.0 software (SPSS Inc., Chicago, Ilinois). The normal distribution of the collected data was confirmed by means of the Kolmogorov-Smirnov test. Demographic and surgical data were compared between groups using the unpaired Student's t-test. Spirometric variables were compared over time between the groups using repeated measures analysis of variance, followed by the Student-Neumann-Keuls post hoc test when necessary. Pain scores and cumulative morphine consumption were analyzed by means of the Friedman test, followed by the Mann-Whitney test when necessary. All data are presented as mean values ± standard deviation, error means, or as otherwise described. The significance level was fixed at 5%. BODY.RESULTS: Patient characteristics and general variables are presented in Table 1. There were no differences between groups with regard to the variables represented in Table 1. Mean surgery duration and CPB length were similar in both groups (392 ± 70 vs. 379 ± 91 minutes) and (98 ± 26 vs. 95 ± 38 minutes) (p=0.606 and p=0.780, respectively). Minimal temperature values were 31.3 ± 2.7°C in the control group and 30.7 ± 2.7°C in the morphine group (p=0.456). All patients achieved hemodynamic stability in the immediate postoperative period, permitting weaning off of the vasoactive drugs within the first 24 hours. The total dose of sufentanil employed was 257.77 μg ± 81.41 in the control group and 200.87 μg ± 55.11 in the morphine group (p= 0.012), and was related to the duration of surgery. Intrathecal morphine did not shorten the postoperative time to extubation between groups (396 ± 234 min in the control group and 349 min ± 175 in the morphine group, p=0.466). None of the patients in our cohort developed respiratory failure after extubation or needed postoperative non-invasive ventilation. All intrathecal punctures were successful at the first or second attempt. No cases of spinal hematoma or treatable pruritus were observed. All patients were discharged from the ICU without postoperative complications. BODY.RESULTS.GAS EXCHANGE AND SPIROMETRIC ANALYSIS: Both group presented a significant decrease in FVC and FEV1 during the first and second postoperative days, with no significant difference between the groups (p=0.068 and 0.085, respectively). The FEV1/FVC ratio did not differ between groups (p=0.68), as presented in Table 2. In ratio did not differ between groups addition, the PaO2/FiO2 at any time point (p >0.05), as presented in Figure 1. BODY.RESULTS.PAIN SCORES, MORPHINE CONSUMPTION, AND PLASMA CONCENTRATIONS: The intensity of pain at rest and with cough was significantly lower in the morphine group at 12, 18, 24, and 36 hours postoperatively when compared to the control group (p <0.05), as shown in panels A and C of Figure 2. With profound inspiration, patients in the morphine group reported pain levels that were significantly less intense at 12, 18, and 24 hours after surgery (panel B of Figure 2). The venous morphine solicitation count was lower in the morphine group at 18 (control group= 108.3 ± 213.8 and morphine group= 20.2 ± 30.3) and 24 hours (control group= 144.4 ± 251.2 and morphine group= 32.4 ± 38.05) (p<0.05). Cumulative morphine consumption was also reduced in the morphine group at 18 (control group= 20.14 ± 17.73 mg and morphine group=14.10 ± 26.15 mg), 24 (control group= 27.82 ± 22.77 mg and morphine group= 13.55 ± 10.49 mg), and 36 hours (control group = 38.50 ± 26.70 and morphine group= 24.30 ± 14.59) postoperatively (Figure 3), as well as upon extubation (control group= 3.32 ± 8.17 mg and morphine group= 0.15 ± 0.49 mg) (p<0.05). Plasma morphine levels were zero at the end of surgery in both groups. However, plasma levels were significantly reduced in the morphine group during the postoperative period 24 hours after the operation (control group= 15.87 ± 18.05 ng mL−1 and morphine group 4.08 ± 5.28 ng mL−1, p=0.029), as presented in Figure 4. Nine patients in the control group and five patients in the morphine group requested rescue analgesia with dipyrone (p= 0.460). BODY.DISCUSSION: In the present study, intrathecal morphine administration in patients undergoing coronary artery bypass graft reduced pain scores, postoperative morphine solicitation, and morphine consumption and its plasma concentration. However, despite better analgesic control, no significant improvements in spirometry or gas exchange were observed. Respiratory system dysfunction is the most prevalent organic complication after cardiac surgery, even in the absence of preoperative pulmonary disease1. Perioperative atelectasis leads to a pulmonary ventilation/perfusion mismatch that likely serves as a major cause of shunt and hypoxemia after cardiopulmonary bypass.14 The impact of postoperative analgesia techniques on pulmonary function outcome has been well studied in non-cardiac surgery, but few studies to date have examined this matter in cardiac surgery.8 A study that enrolled 113 patients submitted for different types of cardiac surgery observed that thoracic epidural analgesia (TEA) combined with general anesthesia followed by patient-controlled thoracic epidural analgesia offered no major advantages with respect to lung function, length of hospital stay, quality of recovery, or morbidity when compared to general anesthesia alone, with both groups followed by patient-controlled analgesia with intravenous morphine.15 In contrast, a meta-analysis including 15 trials enrolling 1.178 patients suggested that TEA significantly reduced the risk of pulmonary complications (OR 0.41), time to tracheal extubation by 4.5 hours, and analog pain scores at rest and with activity.9 In the present study, intrathecal instead of epidural morphine was used because there is no evidence for analgesia superiority between them. However, the risk of spinal hematoma is lower with the intrathecal technique,16 especially with the required anticoagulation prior to cardiac surgery. The last two meta-analyses used to evaluate the use of intrathecal morphine in cardiac surgery did not reveal a correlation between the efficacy of pain control and better respiratory outcome,3,11 likely because these studies did not employ lung function as an end-point11. Some of these studies described no improvement of pain control10 or modest reduction of pain scores,11 with higher doses correlating with better postoperative analgesia17 as well as longer time to extubation.18 In line with the beginning of the fast-track era and its goal to decrease the intubation time after surgery, changes in anesthesia techniques and a reduction in the dose of intrathecal morphine were previously associated with a decreased time to extubation,20 with some cases of prolonged respiratory depression.21 A previous study compared intrathecal morphine doses of 250 and 500 μg and placebo, and showed the superiority of intrathecal analgesia, without alterations in gas exchange19 or an increase in time to extubation.22 In the present study, the selected dose of 400 μg of morphine had no effect on time to tracheal extubation in the postoperative period. According to our results, use of low doses of intrathecal morphine can decrease the intensity of postoperative pain at different times, such as at rest, upon profound inspiration, and upon cough. This suggests that not only does intrathecal morphine provide better analgesia at rest, but also during activities that support walking, respiratory maneuvers, and the use of incentive spirometry. This is important to ensure the quality of patient recovery, since it has been demonstrated that respiratory physiotherapy may reduce rates of pulmonary complications.23–25 Improved analgesia may allow for a decrease in the solicitation and consumption of postoperative analgesics, resulting in lower plasma analgesic levels, as observed in this study. A previous cardiac surgery study that also used intrathecal morphine revealed a venous consumption of 13.5 mg at a dose of 250 μg, and a consumption of 11.7 mg of morphine with an intrathecal dose of 500 μg compared to 21.7 mg in the placebo group 24 hours after the operation.22 In a meta-analysis, 668 patients undergoing cardiac surgery were reported to consume 11 mg of intravenous morphine in intrathecal group .9 The importance of studying the plasma concentration of morphine as a marker for progress in the of treatment of pain was considered by Bonica in 1985, but only a few studies have investigated this idea26 Only one study has been published regarding plasma morphine concentrations during patient control analgesia techniques,27 and no studies reported intrathecal morphine use in the postoperative period or its correlation with pain scores. In patients undergoing hysterectomy with postoperative PCA, an increase in plasma morphine levels resulted in clinical effects 6 to 12 hours after surgery. This lag could be explained by the delay in morphine passing through the hematoencephalic barrier. After this period, a significant reduction in morphine consumption and pain scores was observed, and this can be explained by maintenance of a steady-state level of morphine sufficient to maintain an effective level of analgesia.27 Mutations in the opioid μ receptor were described to support the interindividual variability theory in the population and its possible influences on the use of morphine.28–30 Although the number of patients in our study was insufficient to fully evaluate the adverse effects of intrathecal morphine, no cases of spinal hematoma or treatable pruritus were observed in the present study. Previous studies utilizing doses between 5–24 μg kg−1 for cardiac surgery described a 30% incidence of pruritus,21 which is the most common symptom observed after morphine treatment. Fortunately, this common side effect becomes severe in only 1% of cases.31 The absence of an intrathecal puncture in the control group may have limited our ability to observe adverse effects. Despite the risk of spinal hematoma, the use of intrathecal analgesia in cardiac surgery32 has increased. This increase may be explained by the demand for better analgesia with chest tubes, sternotomy, deambulation, and physiotherapy maneuvers in the absence of a trained pain management team and patient-control pumps. Thus, our study has direct clinical applications. Future studies are being conducted to study the effects of pain control on quality of life, satisfaction, and postoperative recovery, since no additional benefits of intrathecal morphine beyond pain control have been shown until now. None of these quality of life or recovery variables have been correlated with better pain scores, but a modest reduction in satisfaction was shown in the presence of adverse effects.33 Considering the risks of nerve blockade and required anticoagulation in cardiac surgery, further efforts are required to identify better pain control to ensure better patient outcomes.

TITLE: Household costs for personal protection against mosquitoes: secondary outcomes from a randomised controlled trial of dengue prevention in Guerrero state, Mexico ABSTRACT.BACKGROUND: Dengue is a serious public health issue that affects households in endemic areas in terms of health and also economically, imposing costs for prevention and treatment of cases. The Camino Verde cluster-randomised controlled trial in Mexico and Nicaragua assessed the impact of evidence-based community engagement in dengue prevention. The Mexican arm of the trial was conducted in 90 randomly selected communities in three coastal regions of Guerrero State. This study reports an analysis of a secondary outcome of the trial: household use of and expenditure on anti-mosquito products. We examined whether the education and mobilisation activities of the trial motivated people to spend less on anti-mosquito products. ABSTRACT.METHODS: We carried out a household questionnaire survey in the trial communities in 2010 (12,312 households) and 2012 (5349 households in intervention clusters, 5142 households in control clusters), including questions about socio-economic status, self-reported dengue illness, and purchase of and expenditure on insecticide anti-mosquito products in the previous month. We examined expenditures on anti-mosquito products at baseline in relation to social vulnerability and we compared use of and expenditures on these products between intervention and control clusters in 2012. ABSTRACT.RESULTS: In 2010, 44.2% of 12,312 households reported using anti-mosquito products, with a mean expenditure of USD4.61 per month among those who used them. Socially vulnerable households spent less on the products. In 2012, after the intervention, the proportion of households who purchased anti-mosquito products in the last month was significantly lower in intervention clusters (47.8%; 2503/5293) than in control clusters (53.3%; 2707/5079) (difference − 0.05, 95% CIca −0.100 to −0.010). The mean expenditure on the products, among those households who bought them, was USD6.43; 30.4% in the intervention clusters and 36.7% in the control clusters spent more than this (difference − 0.06, 95% CIca −0.12 to −0.01). These expenditures on anti-mosquito products represent 3.3% and 3.8% respectively of monthly household income for the poorest 10% of the population in 2012. ABSTRACT.CONCLUSIONS: The Camino Verde community mobilisation intervention, as well as being effective in reducing dengue infections, was effective in reducing household use of and expenditure on insecticide anti-mosquito products. ABSTRACT.TRIAL REGISTRATION: (ISRCTN27581154). BODY.BACKGROUND: Since its re-emergence in the Americas, dengue has continued to spread in all its clinical forms, despite vector-control efforts on the part of health services from every country in the region, and health services in Latin America use considerable resources to treat cases of dengue fever [1]. In Mexico, the Specific Action Program for Dengue 2013–2018 (Programa de Acción Específico. Prevención y Control de Dengue 2013–2018) confirmed a protocol to identify dengue fever patients as potential carriers of the disease, and to register them in the National Epidemiological Surveillance System (Sistema Nacional de Vigilancia Epidemiológica) to locate cases in time and space. The programme includes guidelines for prevention and control measures to be carried out during visits by health workers to patients' homes and neighboring households, during which a larvicide, temephos, is placed in water containers, and the area surrounding each home is fumigated [2]. Studies in Latin America have estimated the direct and indirect costs of in- and out-patient dengue cases, workdays lost due to the disease, and disability- or quality-adjusted life years [3–7]. Authors have reported on the household costs of actions to prevent mosquito-borne infections in Asia and Africa [8–12]. However, we have not found any published report of a randomised controlled trial of dengue prevention that examined the impact of the trial intervention on household expenditures on prevention. The study reported here describes household expenditures on personal protection measures for dengue prevention and examines the impact on these expenditures as a secondary outcome of the Mexican arm of the Camino Verde trial of community-based activities for dengue prevention, undertaken in Mexico and Nicaragua [13]. Our analysis examines whether Camino Verde's education and mobilisation activities helped to motivate people to control mosquito breeding in their homes and neighbourhoods by non-chemical means and to spend less on personal protection measures. BODY.METHODS: The methods of the Camino Verde trial are described in detail elsewhere [13, 14]. This study is based on findings from the Mexican arm of the trial. The study included a random sample of 90 communities from the most recent census in the three coastal regions in the state of Guerrero, Mexico. After the 2010 baseline household survey, we stratified clusters according to evidence of recent dengue virus infection in children aged 3–9 years and vector indices, and allocated half to receive the intervention. The intervention encouraged community mobilisation using results from the baseline survey. Each intervention cluster adapted the basic intervention - chemical-free prevention of mosquito reproduction - to its own circumstances. However, the government-run dengue control programme, including temephos application to household water containers and fumigation, continued in all clusters. The impact survey of the trial took place in 2012. Both the baseline and the impact survey included a household questionnaire, an entomological survey of larvae and pupae of Aedes aegypti in water containers, and paired saliva samples in children aged 3–9 years to look for evidence of recent dengue infection (doubling of specific IgG levels). The primary trial outcomes included self-reported dengue cases in the past year, serological evidence of recent dengue virus infection, and conventional entomological indices of Aedes aegypti infestation. A secondary outcome measure in the trial was household expenditure on personal protection measures for dengue prevention. BODY.METHODS.ESTIMATION OF PERSONAL PROTECTION EXPENDITURES: The household questionnaire in the baseline and impact surveys included questions about use of anti-mosquito products such as insecticide sprays or spirals, the frequency of their use, and the amount spent on these products during the month before the survey. We estimated the average monthly expenditure per household on insecticides, among households that reported any expenditure on these products during the last month. We also estimated the mean expenditure in the last month across all households, including those who spent nothing. In the surveys the amounts spent were reported in Mexican pesos (MXN). During the years 2010 to 2012 the US dollar (USD) exchange rate for the Mexican dollar fluctuated around MXN13.00 to USD1.00 so we have used that rate for convenience here [15]. Using the data from the baseline survey, we compared the average monthly expenditure on anti-mosquito insecticides between groups of households according to characteristics potentially related to the use of these products: being covered by the healthcare services' temephos (Abate®) distribution programme; reporting at least one case of dengue illness in the previous year; and having evidence of recent dengue infection among children aged 3–9 years. We also examined expenditure on insecticides according to six household social vulnerability characteristics: socioeconomic region; area of residence; ethnicity; household type; education level of the household head; and employment status of the household head. We considered households to be more vulnerable if they were: located in the Costa Chica region; located in rural areas; inhabited by indigenous people; buildings with impermanent construction; headed by someone with a third-grade education or lower; or headed by someone unemployed. BODY.METHODS.EXTRAPOLATION OF EXPENDITURE FIGURES: We extrapolated from expenditures reported by households in the sample in the baseline survey to estimate expenditure on insecticide anti-mosquito products by the whole population of Guerrero State's three coastal regions. We estimated the number of inhabited households per region by dividing the population of the region, from the census by the state average of 4.2 people per household [16]. We applied the proportion of households who reported spending on insecticides in the baseline sample to the estimated number of households in the regions, then calculated the mean total expenditure per region by multiplying by the reported monthly expenditure in the sample households who reported expenditure. To estimate annual expenditure in each region we multiplied the monthly figure by 12. The baseline survey was carried out between January and June 2010, and the reported monthly expenditure in these months would be expected to be relatively low as it is not the main season for mosquitoes. For a more conservative estimate, we multiplied the monthly expenditure by 6, on the assumption that there may be little or no expenditure for six months of the year. BODY.METHODS.ANALYSIS: Trained operators entered data, using EpiData software, with double data entry and validation to minimise keystroke errors. Analysis relied on the public domain software CIETmap [17, 18]. We calculated the mean and standard deviation (SD) for reported monthly household expenditure on insecticides and tested the significance of differences in expenditures between sub-groups using an unpaired t-test or the Kruskal-Wallis non-parametric test for sample difference when variances were different between the groups. We tested the significance of the associations between household reported insecticide use (yes or no) and self-reported dengue cases and serologically-defined dengue infection, using the Mantel-Haenszel procedure and reporting the Odds Ratio (OR) and cluster-adjusted 95% confidence intervals (95% CIca) [19, 20]. From the impact survey, we tested the significance of differences between intervention and control clusters in proportions of households reporting expenditure on insecticides, and in proportions of spending households, and of all households, spending more than the mean amount on these products in the last month, using a cluster t-test. We treated each cluster as a unit in an intention-to-treat analysis (everyone in each cluster, all clusters per allocation) [21]. BODY.RESULTS: In the baseline survey we analysed data from 12,312 households. We excluded 87 of the 12,399 responses (0.7%), because they came from exclusively commercial establishments (stores, workshops, etc.). The impact survey included 10,491 households, 5349 in 45 intervention sites and 5142 in 45 control sites. The reduced number of households in the impact survey was related to the deteriorating security situation in Guerrero State; some people had moved out of the country or to other, more secure, communities. The interviewers encountered more empty houses in the follow up survey than in the baseline. BODY.RESULTS.BASELINE SURVEY.PERSONAL PROTECTION COSTS AT BASELINE: In the baseline survey 44.2% (5433/12,287) of households reported using insecticide anti-mosquito products such as sprays and spirals. The average monthly expenditure for these products was USD 4.86 (SD 4.66). A quarter (24%; 1293/5407) of households reported using anti-mosquito products daily, 20% (1074/5407) used them two or three times a week, 19% (1019/5407) used them once a week, and 37% (2021/5407) used them every two or more weeks. As expected, the reported monthly expenditure varied by reported frequency of use. It was USD6.53 (n = 1281; SD 7.97) for daily use, USD5.35 (n = 1061; SD 4.41) for use two or three times per week, USD4.45 (n = 1007; SD 3.83) for weekly use, and USD3.73 (n = 1990; SD 3.66) for use every two or more weeks. The difference in expenditures between groups with different frequency of use was statistically significant (Kruskal-Wallis H 394; df 3; p < 0.000001). The extrapolation of expenditures from the sample to the whole population of the coastal regions in Guerrero state is shown in Table 1. Expenditure was highest in Acapulco region because of the higher number of households and also the higher proportion of households reporting insecticide use.Table 1Estimated expenditure in USD on insecticides in the three coastal regions of Guerrero State in 2010Acapulco C. Grande C. Chica Estimated number of inhabited households (population/4.2)188,08898,522102,024Estimated number of households using insecticidesa 96,86539,90140,801Amount spent on insecticides in the last month, among those using themb 491,776168,811213,420Amount spent on insecticides in the last yearc 5,901,3122,205,7322,561,040 aThe proportions of households that reported using insecticides in the 2010 baseline study were: Acapulco region 51.5%, Costa Grande region 40.5% and Costa Chica region 40% bThe mean expenditures on insecticides in the last month were: Acapulco USD5.0; Costa Grande USD4.2; and Costa Chica USD5.2. cThe amount shown is calculated by multiplying the monthly amount by 12. A more conservative estimate, assuming the products are not purchased for roughly half the year, multiplies the monthly amount by 6, and gives the following annual expenditures: Acapulco USD2,950,656; C. Grande USD1,012,866; C. Chica USD1,280,520 In the baseline survey, 6.3% of households (780/12,308) reported at least one case of dengue illness in the last 12 months. The rate of self-reported dengue cases was 8.3% (452/5433) among households reporting insecticide use, and 4.8% (327/6850) among households not reporting insecticide use. Households that reported insecticide use were 80% more likely to report a case of dengue illness in the last year than households that did not report insecticide use (OR 1.81; 95% CIca 1.53–2.10). Among households who used insecticide products, the average monthly expenditure was significantly lower among households that did not report any dengue cases in the last year (USD4.80; n = 4905; SD 4.53), than among households that reported one or more cases of dengue illness (USD5.53; n = 448; SD 5.9) (Kruskal-Wallis H 4.4; df 1; p = 0.03). Some 10.5% (1284/12,251) of households reported that government workers had never placed temephos in their water containers; 33% (4063/12,251) of households reported receiving temephos in the last month; 53% (6566/12,251) received temephos two months ago or more; and 3% (338/12,251) could not specify when they received temephos. In households which had never been covered by the temephos programme, 36% (461/1284) reported using insecticide anti-mosquito products, while among those that had received temephos at least once the rate was 45% (4955/10,967). Households which were not covered by the temephos programme were 32% less likely to have used insecticide products compared with those which were covered by the programme (OR =0.68; 95%CIca 0.55–0.84). Among households using insecticide products, the average monthly expenditure was not significantly different between those with temephos coverage (4.9 USD; n = 4888; SD 4.6) and those without it (4.9 USD; n = 449; SD 5.1; Kruskal-Wallis H 0.968; df1; p = 0.33). BODY.RESULTS.BASELINE SURVEY.EXPENDITURES AND SOCIAL VULNERABILITY: There were significant differences in the average monthly expenditure on insecticides based on household social vulnerability characteristics (Table 2). Expenditures were higher in Acapulco region than in the other two regions. Expenditures were generally lower among more vulnerable households. Rural households and those with a non-permanent construction spent less than urban households or those with a permanent construction. Households with a less educated head or an unemployed head spent less than those with a more educated head or an employed head.Table 2Average monthly household expenditure on insecticides in USD by social vulnerability characteristics in 2010 baseline surveyCharacteristicn=% of households using insecticidesMean expenditure last monthSDp= Acapulco218951.55.14.8<0.0000001 Costa Grande161840.54.23.9 Costa Chica155140.05.25.2 Rural265540.44.64.4<0.0000001 Urban270348.75.14.9 Indigenous25356.55.14.40.21 Mestizos 509243.74.94.7 Non-permanent house62636.54.54.10.00002 Semi-permanent house210441.64.74.8 Permanent house261049.25.04.7Household head education: Less than 3rd grade165138.14.54.5<0.0000001 4th grade to high-school315646.44.94.7 Technical school or higher49956.15.65.2 Household head unemployed77640.34.64.90.00009 Household head employed457045.04.94.6 BODY.RESULTS.IMPACT OF THE TRIAL INTERVENTION: Table 3 shows the proportions of households reporting purchase of anti-mosquito products in the last one month and the mean expenditure on these products: among the households who bought them, and among all households. In both the intervention and control groups the proportion of households buying anti-mosquito products and the amount they spent on them are higher than in the baseline survey (see Table 2), reflecting the timing of the surveys: the 2010 baseline took place in the "low season" for mosquitos (January to May 2010), and the 2012 impact survey took place in the "high season" for mosquitos in August–November.Table 3Proportion of households that purchased anti-mosquito products, and expenditure during the last month among those who purchased the products, in trial intervention and control sites surveyed in August–November 2012Intervention clustersControl clustersDifference of proportions (95%CIca)Surveyed households53495142Proportion of households that purchased anti-mosquito productsa 47.8% (2530/5293)53.3% (2707/5079)−0.05 (−0.1 to −0.01) Among households spending anything Mean expenditure in the last month (USD)6.0 (SD 5.9)6.83 (SD 6.84)Proportion spending more than the mean of USD 6.43b 30.4% (768/2530)36.7% (993/2707)−0.06 (−0.12 to −0.01) Among all households Mean expenditure in the last month (USD)2.86 (SD 5.12)3.65 (SD 6.04)Proportion spending more than the mean of USD 3.25c 30.6% (1622/5293)37.5% (1906/5079)−0.07 (−0.09 to −0.05) aCluster t-test. t = −2.193, df 88 p = 0.031 bCluster t-test. t = −1.978, df 88, p = 0.05 cCluster t-test. t = −2.653, df 88, p = 0.009 Table 3 shows that after the intervention, the proportion of households that purchased anti-mosquito products in the last month was significantly lower in the intervention sites (48%) than in the control sites (53%). At the time of the 2010 baseline, 43.2% (2647/6130) of households in clusters that subsequently received the intervention used insecticide anti-mosquito products, compared with 45.2% in clusters that subsequently served as controls; the difference was not statistically significant (cluster t-test, t = 0.515, p = 0.608). In 2012, the mean monthly expenditure on these products, among households that purchased them, was lower in the intervention sites than in the control sites, and the proportion spending more than the mean was significantly lower in the intervention sites (Table 3). Also in 2012, the mean monthly expenditure on anti-mosquito products across all households (including those not spending anything) was lower in the intervention sites, and the proportion spending more than this mean amount was significantly lower in the intervention sites (Table 3). BODY.DISCUSSION: Our study shows that expenses for the purchase of products for personal protection against mosquitoes are an important proportion of monthly household incomes in Guerrero state. According to the 2012 National Household Income and Expenditure Survey, the average monthly income for Mexican households in the lowest income decile was USD171 [22]. The monthly expenditures on insecticide anti-mosquito products reported in our 2012 impact survey, of USD6.0 in intervention communities and USD6.83 in reference communities, represent 3.3% and 3.8% respectively of monthly income for people in this decile. The findings from our study add to the existing literature on household expenditures on anti-mosquito products, as a means of protection against dengue and other mosquito-borne diseases. Mulla and colleagues estimated an expenditure between USD13.75 and USD86.13 on anti-mosquito products per household per year in four communities in Thailand, and reported that these expenses represented between 0.3% and 0.7% of the annual household income in Thailand [8]. A 2003 study in The Gambia reported that most (81%) of the recurring household expenditure for malaria protection was on insecticide anti-mosquito products rather than on bed nets [23]. Another 2003 survey in the Pondicherry region of Southern India reported that 99% of urban dwellers and 73% of rural dwellers used insecticide anti-mosquito products at some time in the year, and that annual expenditure on these products in urban areas was 0.63% of annual per capita income [9]. Similarly, a survey in Jaffna district, Sri Lanka, reported that 96% of respondents spent funds on products for personal protection against mosquitoes, mainly spirals, with monthly expenditure between USD0.70 and USD12.53 [10]. A survey in Orissa, India, reported use of anti-mosquito products by 99% of urban and 84% of rural households, with an average monthly expenditure of USD8.13 in urban areas and USD5.90 in rural areas [11]. In north-eastern Tanzania, a survey reported that households spent an average of USD0.18 on bed nets and their treatment each fortnight (47% of total prevention costs) and USD0.21 on insecticide anti-mosquito products (50% of the total) [12]. A 2012 survey of household incomes and expenditures in Mexico reported a household quarterly expenditure of USD21.83 (USD7.28 per month) on insecticide anti-mosquito products [22]. Table 4 summarises the monthly expenditure on anti-mosquito products reported by other authors in other countries. Using a Purchasing Power Parity conversion factor [24], the monthly expenditure estimates in our study fit within the range of expenditures previously reported.Table 4Summary of monthly household expenditures on anti-mosquito products reported by other authorsAuthor, country and yearMonthly expenditure reported (USD)Equivalent in 2012 USDPurchasing Power Parity conversion factorPurchasing Power Parity expenditureMulla et al. Thailand 1999 [8]4.00–25.005.50–34.450.413.75–86.13Wiseman et al. Gambia 2003 [23]2.503.130.310.43Surendran et al. Sri Lanka 2007 [10]0.19–3.400.21–3.760.30.70–12.53Babu et al. India 2007 [11]1.60–2.201.77–2.440.35.90–8.13McElroy et al. Tanzania 2009 [12]0.420.440.41.10ENIGH, Mexico 2012 [22]7.287.280.612.13 In the baseline survey, we found an association between a self-reported case of dengue illness in the household and a greater likelihood of the household purchasing insecticide anti-mosquito products. We have to be cautious in interpreting this finding from a cross-sectional enquiry. It could be that the response of the health services to a case of dengue, which includes placing temephos into water containers in the index household and surrounding households, as well as fumigation of the area, encourages the residents to use more anti-mosquito products. Our finding of more expenditure on insecticides with more education of the household head (see Table 2) runs contrary to the idea that more educated households would be more aware of the health dangers of insecticides so would use them less. However, there is little concern about toxicity of insecticide products in Mexico [25] and the higher expenditure on such products when the household head is educated probably reflects the better economic status of such households. Other authors have also reported more expenditure on insecticide products when the household head is more educated [12]. A small study in Sri Lanka found no association between level of education and awareness about mosquito-borne diseases [26]. As described in the main report of the Camino Verde trial, there was a small but significant increase in knowledge of the dengue vector related to the intervention; but the impact survey questionnaire did not ask about knowledge of health effects of insecticides [13]. A key aim of our study was to estimate the impact of the Camino Verde trial on a stated secondary outcome of the trial: use of and expenditure on insecticide anti-mosquito products. Our findings indicate that after the trial intervention, fewer households in the intervention clusters purchased insecticide anti-mosquito products, and those who did buy them spent less on them than did households in the control clusters. Discouraging the use of insecticide sprays or spirals as protection against mosquitoes and the illnesses they are associated with, such as dengue, was not an explicit activity in the intervention design. However, community educators, called brigadistas, encouraged reflection and dialogue about various options for controlling mosquitoes, and the emphasis in the educational messages, which involved showing householders where mosquito larvae and pupae were lurking on their own premises and explaining to them how the mosquito development cycle could be interrupted, was on non-chemical solutions. The cost implications of the brigadistas' interventions and discussions with householders are considered in another article about the Camino Verde trial [27]. Our finding that vulnerable households spent less on insecticides for personal protection against mosquitoes (see Table 2) could suggest that these personal protection measures might be too expensive for the poorest section of the population. Non-chemical control methods are accessible to all households, and are an effective means of prevention [28]. BODY.CONCLUSION: The evidence from this study supports the hypothesis that the participatory intervention of Camino Verde, based on bringing the community voice to action for dengue prevention, can lead to a more sustainable physical and biological control of the Aedes aegypti vector, with a lower number of houses purchasing anti-mosquito products and less expenditure on these products. Added benefits of reduced reliance on insecticides for mosquito control are a reduction in their potential to harm human health and the environment. The household resources saved can be available to meet other household needs.

TITLE: A Randomized, Open-Label Trial to Evaluate Switching to Elvitegravir/Cobicistat/Emtricitabine/Tenofovir Alafenamide Plus Darunavir in Treatment-Experienced HIV-1-Infected Adults ABSTRACT: Supplemental Digital Content is Available in the Text. BODY.INTRODUCTION: Morbidity and mortality in HIV infection have dramatically improved after the introduction of antiretroviral therapy (ART),1–3 even among patients with extensive treatment experience and multiclass drug resistance.4 With the approval of new drugs, particularly the integrase strand transfer inhibitors (INSTIs) and more potent protease inhibitors (PIs) [ie, darunavir (DRV)], construction of fully suppressive "salvage" regimens, containing at least 2 effective drugs from different classes, was possible.5 Despite these advances, "salvage" regimens are often complicated regimens with high pill burden, high dosing frequency, and/or dietary restrictions, factors resulting in higher cost, more side effects, poorer quality of life, and greater risk for nonadherence that may lead to virologic failure and accumulation of additional drug resistance.4,6 Current treatment guidelines recommend regimen simplification to maintain viral suppression if future treatment options are not compromised,7,8 but simplification is challenging in treatment-experienced patients with a history of drug resistance. Newer combination tablets enable simplification of these regimens; however, concerns of potential drug interactions in the absence of pharmacokinetic (PK) and pharmacodynamic data have limited their use.9 The single-tablet coformulation of elvitegravir/cobicistat/emtricitabine/tenofovir alafenamide (E/C/F/TAF) has demonstrated high efficacy and improved renal and bone safety compared with tenofovir disoproxil fumarate (TDF)-containing regimens in phase 3 clinical trials of HIV-infected participants.10–12 E/C/F/TAF has been approved by the United States (US) Food and Drug Administration (FDA), European Medicines Agency, and other health authorities for treatment of naïve and stably suppressed patients age 12 and older and is one of the recommended initial regimens in guidelines in the USA and in Europe.13–17 DRV is a recommended protease inhibitor with a high genetic barrier to resistance, well-established safety profile at a once-daily dose in treatment-experienced patients without DRV-associated resistance mutations, and can be boosted by either ritonavir or cobicistat.18,19 The 2-tablet, once-daily combination of E/C/F/TAF plus DRV may be an effective treatment option in select treatment-experienced patients with multiclass resistance. The goal of our phase 3, open-label, randomized trial (GS-US-292-0119) was to evaluate the efficacy, safety, and PK parameters of switching virologically suppressed participants with a history of treatment failure to a 2-tablet, once-daily antiretroviral regimen combining E/C/F/TAF plus DRV. We report results though week 48. BODY.METHODS.STUDY DESIGN AND PARTICIPANTS: We enrolled participants into this phase 3, open-label, randomized trial at 62 academic, private practice, and community health centers in the USA and Canada between September 2013 and September 2014 (Study Protocol available in Supplemental Digital Content 1, http://links.lww.com/QAI/A932). Eligible participants were HIV-infected adults (aged ≥18 years) who were virologically suppressed (plasma HIV-1 RNA <50 copies per milliliter) on a current antiretroviral regimen containing DRV boosted by ritonavir (600/100 mg twice daily or 800/100 mg once daily) continuously for at least 4 months before screening. Treatment history included failing at least 2 prior antiretroviral regimens and confirmed resistance by historical genotype to at least 2 different classes of antiretrovirals. Participants must have had no history of integrase inhibitor resistance or be INSTI naive, or be currently suppressed on the following INSTIs [raltegravir, elvitegravir, or dolutegravir (50 mg once daily, but not twice daily)]. We allowed a history of up to 3 thymidine analogue mutations and/or K65R, but no history of Q151M or T69 insertion mutations. Eligible participants could not have any DRV-associated resistance mutations, including V11I, V32I, L33F, I47V, I50V, I54L/M T74P, L76V, I84V, and L89V. The Stanford HIVdb algorithm v8.01 was used to calculate genotypic susceptibility scores (GSS). For each drug, a 5-point scale was used: susceptible, potential low-level resistance, low-level resistance, intermediate-level resistance, and high-level resistance were scored as 1, 0.75, 0.5, 0.25, and 0, respectively. The total genotypic susceptibility scores for a given regimen were calculated as the sum of the scores for each individual drug. Participants had an estimated glomerular filtration rate (eGFR) by the Cockcroft-Gault formula of at least 50 mL/min. We randomized participants in a 2:1 ratio using an interactive voice/web response system to switch to coformulated E/C/F/TAF 150/150/200/10 mg (Gilead Sciences, Foster City, CA) plus DRV 800 mg (Janssen) once daily or to stay on their baseline regimen. E/C/F/TAF plus DRV was supplied by Gilead; participants who continued their baseline regimen obtained treatment by prescription. This study was conducted in accordance with the Declaration of Helsinki. Central or site-specific institutional review boards or ethics committees reviewed and approved the protocol. We obtained written informed consent from all participants before screening. This study was registered with ClinicalTrials.gov (Identifier NCT01968551). BODY.METHODS.PROCEDURES: Randomized participants were seen at screening, baseline, weeks 2, 4, 8, 12, 16, 24, 36, 48, and every 12 weeks thereafter. Laboratory tests included hematological analysis, serum chemistry tests, fasting lipid parameters, CD4 counts, measures of renal function [eGFR by Cockcroft-Gault, urine protein-to-creatinine ratio, retinol binding protein-to-creatinine ratio, beta-2-microglobulin-to-creatinine ratio (Covance Laboratories, Indianapolis, IN)], and measurement of HIV RNA concentration (Roche TaqMan 2.0; Roche Diagnostics, Rotkreuz, Switzerland). Fasting lipid parameters were not measured in this study. Participants with confirmed virologic rebound (HIV-1 RNA ≥400 copies per milliliter) had confirmatory samples sent for resistance analysis by GeneSeq Integrase, PhenoSense GT, and PhenoSense Integrase (Monogram Biosciences, South San Francisco, CA). Patient-reported outcomes included validated instruments such as visual analog scale adherence and HIV-treatment satisfaction questionnaires.20–22 For those receiving E/C/F/TAF plus DRV who agreed to participate, we performed an intensive PK substudy (sample collection predose up to 24 hours postdose at weeks 2, 4, or 8) to assess plasma concentrations of select analytes using fully validated high-performance liquid chromatography–tandem mass spectroscopy bioanalytical methods (Quest Pharmaceutical Services LLC, Newark, DE). BODY.METHODS.STATISTICAL ANALYSIS: The primary efficacy endpoint was the percentage of participants with HIV-1 RNA <50 copies per milliliter at week 24 using the US FDA-defined snapshot algorithm23,24 using the full analysis set (all randomized participants who received at least one dose of study drug). We planned to randomize 100 participants to E/C/F/TAF plus DRV and 50 to remain on their baseline regimen, resulting in 53% power to show switching to E/C/F/TAF plus DRV was noninferior to remaining on baseline regimens with respect to the primary endpoint. We assessed noninferiority of E/C/F/TAF plus DRV vs. baseline regimens using a 2-sided exact 95% confidence interval (CI) approach (alpha level of 0.025), with a prespecified margin of 12%. Because an alpha of 0.00001 was spent on interim analyses at week 12, the significance level for the 2-sided test was adjusted to 0.04999 (corresponding to 95.001% CI). We estimated the exact CI based on unconditional exact methods using 2 inverted one-sided tests with the standardized statistic and calculated the P-value using Fisher's exact test. We also performed a secondary per-protocol analysis (full analysis set excluding those who had any major protocol violation). Secondary efficacy endpoints included the percentage of participants with HIV-1 RNA <50 copies per milliliter at week 48 using the US FDA-defined snapshot algorithm and the change from baseline in CD4 cell count at weeks 24 and 48. We conducted prespecified subgroup analyses for virologic response at weeks 24 and 48 to assess treatment differences by age, sex, and race. We used descriptive statistics for the safety analysis set, which included all who received at least one dose of study drug. Adverse events were coded with the Medical Dictionary for Regulatory Activities (version 18.0). We evaluated change from baseline in eGFR (Cockcroft-Gault), retinol binding protein-to-creatinine ratio, and beta 2 microglobulin-to-creatinine ratio and used the 2-sided Wilcoxon rank-sum test to compare the 2 groups. We compared percent change from baseline in urine protein-to-creatinine ratio between the 2 groups, adjusting for baseline value using rank analysis of covariance. For the PK analyses, we used descriptive statistics to summarize plasma concentrations and calculated geometric mean (95% CI) and mean (SD) of select natural log-transformed PK parameters. We compared HIV treatment satisfaction total scores at weeks 24 and 48 between the 2 groups using an ANCOVA model, adjusting for baseline score. BODY.RESULTS: Of the 199 screened participants, 135 enrolled and received at least one dose of study drug (89 randomized to switch to E/C/F/TAF plus DRV, 46 randomized to remain on their baseline regimen) (Fig. 1). Fewer participants in the E/C/F/TAF plus DRV group prematurely discontinued study drug compared with the baseline regimen group (2% vs. 11%). FIGURE 1.Consort flow diagram. Baseline demographic and antiretroviral regimen characteristics are shown in Table 1. Although overall most participants were men (75%) and white (50%), women (25%) and black (45%) participants were well represented. Compared with the baseline regimen group, more men (E/C/F/TAF plus DRV, 82% vs. baseline regimen, 61%) and fewer black participants (E/C/F/TAF plus DRV, 39% vs. baseline regimen, 57%) were randomized to the E/C/F/TAF plus DRV group. The median age was 49 years (range, 23–70 years). Comorbid conditions included hyperlipidemia (40%), hypertension (35%), diabetes mellitus (9%), and cardiovascular disease (6%). At baseline, participants were taking a median of 5 tablets per day (range, 2–10 tablets per day). Thirty-nine percent of participants were taking 6 or more tablets per day; 65% were taking at least one antiretroviral medication twice daily with 37% taking DRV twice daily. Overall, 59% of participants were on a TDF-based regimen, 11% on an abacavir-based regimen, and 57% on an integrase inhibitor. The distribution of genotypic susceptibility scores at study entry was similar across treatment groups. TABLE 1. Baseline Demographic and Antiretroviral Regimen Characteristics All participants had at least 2-class genotypic resistance per eligibility criteria, the most prevalent being M184V/I for 95% of participants with nucleoside reverse transcriptase inhibitor mutations and K103N/S for 88% of participants with nonnucleoside reverse transcriptase inhibitor resistance-associated mutations (Supplemental Digital Content, Table 1, http://links.lww.com/QAI/A932). The tenofovir signature mutation K65R was present at 20% and 30% in the E/C/F/TAF plus DRV and baseline regimen groups. Participants with thymidine analogue mutations (up to 3) represented 44% and 39% of the E/C/F/TAF plus DRV and baseline regimen groups, respectively (Table 1). BODY.RESULTS.PHARMACOKINETICS: Fifteen participants who received E/C/F/TAF plus DRV participated in the PK substudy. Mean steady-state trough plasma concentrations (Ctrough) for elvitegravir (464 ng/mL) and DRV (1250 ng/mL) were >10-fold and >22-fold higher than the protein-adjusted IC95 value of elvitegravir (45 ng/mL) and protein adjusted EC50 value for DRV for virus with no DRV resistance-associated mutations (55 ng/mL), respectively (Fig. 2). Plasma exposure of TAF (mean AUC of 89.9 ng·h·mL−1) was within the range of safe and efficacious TAF exposure established in the phase 3 pivotal E/C/F/TAF studies (mean AUCt 47.2–1869.3 ng·h·mL−1). Consistent with findings from the phase 3 pivotal E/C/F/TAF vs. E/C/F/TDF studies, the plasma tenofovir exposure in the current study after administration of E/C/F/TAF plus DRV (mean AUC: 367 ng·h·mL−1) was markedly lower than the tenofovir exposure after administration of E/C/F/TDF. The primary PK parameters for elvitegravir, DRV, cobicistat, TAF, and its metabolite tenofovir are presented in Supplemental Digital Content, Table 2, http://links.lww.com/QAI/A932. FIGURE 2.Pharmacokinetic substudy results (N = 15). BODY.RESULTS.EFFICACY: E/C/F/TAF plus DRV was noninferior to baseline regimens for the primary outcome (HIV-1 RNA <50 copies per mililiter at week 24; FDA-snapshot algorithm) using the full analysis set (96.6% vs. 91.3%; difference 5.3%; 95.001% CI: −3.4% to 17.4%) (Fig. 3). Virologic success rates at week 24 using the per-protocol analysis set were consistent with the full analysis set (97.6% vs. 100.0%; difference −2.4%; 95.001% CI: −8.6% to 5.9%). At week 48, E/C/F/TAF plus DRV met the prespecified criteria for noninferiority and superiority using the full analysis set (94.4% vs. 76.1%, difference 18.3%; 95.001% CI: 3.5% to 33.0%) (Fig. 2), with consistent results using the per-protocol analysis set (97.6% vs. 85.4%; difference 12.3%; 95.001% CI: 1.8% to 26.4%). Through week 48, no participants discontinued because of lack of efficacy. Seven participants discontinued (E/C/F/TAF plus DRV, n = 2; baseline regimen, n = 5) because of investigator's discretion (n = 1 on E/C/F/TAF plus DRV), lost to follow-up (n = 2 on baseline regimen), or withdrawal of consent (n = 4; 1 on E/C/F/TAF plus DRV, 3 on baseline regimen). Virologic failure and treatment discontinuation (because of withdrawal of consent or loss to follow-up) contributed to the lower percentage of virologic success in the baseline regimen group, with each factor accounting for 11% of participants. CD4 cell counts remained stable through week 48 in both groups, with a mean (SD) change from baseline at week 48 of 5 (162.6) cells per microliter for E/C/F/TAF plus DRV and 41 (104.2) cells per microliter for baseline regimens (P = 0.21). FIGURE 3.Virologic outcome at weeks 24 and 48. At week 48, all virologic failure was due to plasma HIV-1 RNA ≥ 50 copies/mL. One participant in the baseline regimens group had confirmed viral rebound (HIV-1 RNA > 400 copies/mL). The remaining participants had subsequent plasma HIV-1 RNA < 50 copies/mL. No differences in virologic success at week 24 were seen in the predefined subgroups of age, sex, and race; however, at week 48, virologic success was higher for the E/C/F/TAF plus DRV-treated participants aged <50 years (difference 22.5%, 95% CI: 3.6% to 43.6%) and male (difference 15.9%, 95% CI: 1.5% to 35.0%) (Supplemental Digital Content, Fig. 1, http://links.lww.com/QAI/A932). BODY.RESULTS.RESISTANCE: Because no participants in the E/C/F/TAF plus DRV group had confirmed virologic rebound with HIV-1 RNA >400 copies per milliliter through week 48, none were tested for resistance. One participant in the baseline regimen group, on raltegravir, ritonavir-boosted DRV, and etravirine, had viral rebound (week 36) and was tested for resistance. Historical genotyping confirmed 2-class resistance, with PR (L10V, M361) and RT (D67N, K70R, K103N, Y181I, T215Y) mutations. Resistance results showed newly detected resistance mutations (K65R and M184V); these resistance mutations were likely preexisting as this participant had a history of treatment with TDF and emtricitabine. BODY.RESULTS.SAFETY: Participants in the E/C/F/TAF plus DRV group had a higher rate of any adverse events [92% (n = 82) vs. 78% (n = 36)]. Most adverse events were mild or moderate in severity, and no participants discontinued study drug because of an adverse event. The most commonly reported adverse events (≥5%) were back pain and upper respiratory tract infection [10% (n = 9) each] and bronchitis [8% (n = 7)] for E/C/F/TAF plus DRV and diarrhea [9% (n = 4)] and back pain, cough, influenza-like illness, musculoskeletal pain, and sinusitis [7% (n = 3) each] for baseline regimens. Ten percent of participants (n = 9) in the E/C/F/TAF plus DRV and 2% (n = 1) in the baseline regimen group had a serious adverse event (Supplemental Digital Content, Table 3, http://links.lww.com/QAI/A932), none of which was related to study drug. A similar percentage of participants in each treatment group had grade 3 or 4 laboratory abnormalities [E/C/F/TAF plus DRV, 11% (n = 10); baseline regimens, 9% (n = 4)]. No treatment-related deaths occurred; 1 participant (E/C/F/TAF plus DRV group) with diffuse large B cell lymphoma died of sepsis. Two participants (2%) in the E/C/F/TAF plus DRV group and 1 participant (2%) in the baseline regimen group had traumatic fractures, considered unrelated to study drug. BODY.RESULTS.RENAL SAFETY: No participants had any serious renal adverse event nor discontinued study drugs because of a renal adverse event; no participant developed proximal renal tubulopathy. There were no differences between the 2 groups in median changes from baseline in eGFR (week 48: +7.4 mL/min vs. +3.9 mL/min; P = 0.18). Participants in the E/C/F/TAF plus DRV group had statistically significant declines in both measures of quantitative proteinuria (urine protein-to-creatinine ratio) and tubular proteinuria (retinol binding protein-to-creatinine ratio and beta 2 microglobulin-to-creatinine ratio) at week 48 vs. increases in the baseline regimen group (Fig. 4). FIGURE 4.Renal safety: urine protein-to-creatinine ratios. Differences between treatment groups in changes in quantitative total proteinuria (UPCR) and in specific markers of proximal tubular proteinuria (RBP:Cr and β-2M:Cr) were statistically significant, favoring E/C/F/TAF + DRV. BODY.RESULTS.PATIENT-REPORTED OUTCOMES: Although both arms showed improved patient-reported outcome scores, participants in the E/C/F/TAF plus DRV group reported statistically significantly higher mean HIV treatment satisfaction total scores at weeks 24 and 48 (Fig. 5) and answered questions specific to the flexibility or convenience of and the patient's satisfaction with the treatment more favorably. Based on the visual analog scale adherence questionnaire, participants on E/C/F/TAF plus DRV consistently reported fewer days with missed doses, whereas those on their baseline regimen reported a higher number of days with missed doses as the study progressed: more participants in the E/C/F/TAF plus DRV group reported <2 days with missed doses in the 30 days before week 24 (90% vs. 74%) or 48 (86% vs. 59%) and no days with missed doses in the 4 days before weeks 24 (98% vs. 83%) or 48 (99% vs. 73%). FIGURE 5.Overall HIV treatment satisfaction (HIV-TSQ). aANCOVA HIV-TSQ at baseline 0–6 response range, n = 10 questions, higher score = better satisfaction in recent weeks; HIV-TSQ (change) post-baseline: −3–3 response range, higher–better satisfaction. bFrom ANCOVA model (treatment as fixed effect; baseline total HIV-TSQ score as covariate). BODY.DISCUSSION: This was the first randomized trial of a simplified, 2-tablet, once-daily regimen combining a boosted protease inhibitor with an integrase inhibitor and 2 nucleoside reverse transcriptase inhibitors in virologically suppressed, treatment-experienced adults with 2-class to 3-class drug resistance. Overall, steady-state plasma exposure for the components of E/C/F/TAF and DRV was within the safe and efficacious ranges of these analytes. Importantly, PK results of this study demonstrated that the pharmacodynamic ("boosting") effect of cobicistat on elvitegravir and DRV was maintained upon coadministration of E/C/F/TAF plus DRV, yielding trough concentration levels 10-fold to 22-fold greater than inhibitory and effective concentration thresholds for adequate elvitegravir and DRV antiviral activity. Treatment with E/C/F/TAF plus DRV resulted in high rates of virologic suppression. E/C/F/TAF plus DRV was noninferior to continuing on baseline regimens at week 24 and met prespecified criteria for noninferiority and superiority at week 48. No participant discontinued because of lack of efficacy. Virologic failure and treatment discontinuation (because of withdrawal of consent or loss to follow-up) contributed to the lower percentage of virologic success in the baseline regimen group. At week 48, E/C/F/TAF plus DRV was superior for the subgroups of participants either aged <50 years or male; more men were randomized to the E/C/F/TAF plus DRV group than to the baseline regimen group. No new virologic resistance was detected among participants who switched to E/C/F/TAF plus DRV. Treatment with E/C/F/TAF plus DRV was well tolerated. Participants in the E/C/F/TAF plus DRV group reported a higher rate of adverse events, which was not unexpected for individuals initiating a novel therapy in comparison with those continuing their well-tolerated baseline regimen in an open-label study. Indeed, as assessed by a validated survey tool, although treatment satisfaction was evenly matched between randomized groups at baseline, satisfaction improved among participants who switched to E/C/F/TAF plus DRV. This observation underscores that, for a population with extensive ART experience including prior complicated regimens, there is an unmet need for more convenient treatment options. Simplified regimens improve adherence, as demonstrated in the current study in which participants who switched to E/C/F/TAF plus DRV consistently reporting fewer days with missed doses and greater virologic success at 48 weeks than those remaining on their baseline regimens. Long-term safety considerations are an important element in antiretroviral simplification strategies. Proteinuria and specifically proximal tubular proteinuria have been shown to increase risk of mortality or cardiovascular events in both the general population and in HIV-infected individuals.25–27 Upon entry into the current study, most participants were receiving regimens containing TDF. Switching to a regimen containing TAF improved glomerular and tubular function, including decreases in overall and tubular proteinuria, consistent with results of other TDF to TAF switch studies.11,12,28 Tenofovir (but not TAF) is actively transported from the blood into renal proximal tubule cells by the organic anion transporters (OAT) 1 and OAT3, resulting in tenofovir accumulation in the proximal tubule.29,30 The marked (>90%) reduction in circulating tenofovir after switching to TAF is likely the reason for the improvements in clinical renal toxicity compared with continuing on TDF.10 Participants in our study were representative of the treatment-experienced population who started treatment before the HAART era, with a median age of 49 years, approximately 15 years older than in treatment-naive trials,10 and high rates of comorbid conditions, including hyperlipidemia, hypertension, diabetes mellitus, and cardiovascular disease. This improved renal safety profile is important in a population of treatment-experienced individuals, who are likely to be older and have more cardiovascular and renal risk factors than a naive population. Limitations to this study include the open-label study design; as such, between-group differences in adverse events and subjective safety reports should be interpreted with caution. More robust reporting of adverse events for the experimental arm (E/C/F/TAF plus DRV) may have occurred in this open-label trial. In addition, approximately one-fourth of participants who switched to E/C/F/TAF plus DRV were newly exposed to a new drug class (INSTI), which may have also contributed to additional reporting of adverse events in this group. Another limitation of our study is the relatively small sample size and hence the inability to control for potential imbalance of important factors, such as comorbidities, impacting safety outcomes between the 2 arms. Participants who switched to E/C/F/TAF plus DRV received drugs provided by Gilead; those who continued their baseline regimen obtained treatment by prescription. This difference in study drug supply might have contributed to the difference in the rates of early discontinuation of study drugs. Because this study excluded participants with DRV mutations or more than 3 thymidine analogue mutations, results should not be extrapolated to such individuals. Our study provides important guidance for the management of treatment-experienced individuals with multiclass resistance who have traditionally required multitablet "salvage" regimens to maintain virologic suppression. Single-tablet regimens have generally been options only for treatment-naive individuals or those switching therapy without a history of prior resistance. Strategic regimen simplification to the 2-tablet, once daily combination E/C/F/TAF plus DRV was noninferior at 24 weeks and virologically superior at 48 weeks, to remaining on baseline regimens, and was associated with greater patient satisfaction and adherence. BODY.SUPPLEMENTARY MATERIAL: SUPPLEMENTARY MATERIAL

TITLE: Phase I Randomized Safety Study of Twice Daily Dosing of Acidform Vaginal Gel: Candidate Antimicrobial Contraceptive ABSTRACT.BACKGROUND: Acidform gel, an acid-buffering product that inactivates spermatozoa, may be an effective topical non-hormonal contraceptive. This study was designed to evaluate the safety of vaginal dosing and effects of Acidform on mucosal immune mediators, antimicrobial properties of genital secretions, and vaginal microbiota. ABSTRACT.METHODS: Thirty-six sexually abstinent U.S. women were randomized to apply Acidform or hydroxyethylcellulose (HEC) placebo gel twice daily for 14 consecutive days. Safety was assessed by symptoms and pelvic examination. The impact of gel on mucosal immunity was assessed by quantifying cytokines, chemokines, antimicrobial proteins and antimicrobial activity of genital secretions collected by cervicovaginal lavage (CVL) at screening, 2 hours after gel application, and on days 7, 14 and 21. Vaginal microbiota was characterized at enrollment and day 14 using species-specific quantitative PCR assays. ABSTRACT.RESULTS: The median vaginal and cervical pH was significantly lower 2 hours after application of Acidform and was associated with an increase in the bactericidal activity of CVL against E. coli. However, 65% of women who received Acidform had at least one local adverse event compared with 11% who received placebo (p = 0.002). While there was no increase in inflammatory cytokines or chemokines, CVL concentrations of lactoferrin and interleukin-1 receptor antagonist (IL-1ra), an anti-inflammatory protein, were significantly lower following Acidform compared to HEC placebo gel application. There were no significant changes in Lactobacillus crispatus or Lactobacillus jensenii in either group but there was a decrease in Gardnerella vaginalis in the Acidform group (p = 0.08). ABSTRACT.CONCLUSIONS: Acidform gel may augment mucosal defense as evidenced by an increase in bactericidal activity of genital secretions against E. coli and a decrease in Gardnerella vaginalis colonization. However, Acidform was associated with more irritation than placebo and lower levels of antimicrobial (lactoferrin) and anti-inflammatory (IL-1ra) proteins. These findings indicate the need for additional safety studies of this candidate non-hormonal contraceptive. ABSTRACT.TRIAL REGISTRATION: ClinicalTrials.gov NCT00850837 BODY.INTRODUCTION: Several epidemiological studies indicate that systemic hormonal contraception, particularly progesterone-containing injectables, may be associated with an increased risk of both HIV acquisition and transmission [1]–[3]. Moreover, nonoxynol-9 (N-9), approved in the United States as a vaginal contraceptive, provides no protection against HIV or other sexually transmitted infections [4], [5] and frequent use was shown to be associated with an increased risk of HIV acquisition [6]. Thus, the development of safe and effective alternative contraceptives is a major global health priority. The healthy human vagina in reproductive aged women is acidic, with a pH ranging from 3.5 to 4.5, primarily because of lactic acid and this environment inactivates sperm [7]. However, following sex, the pH is neutralized to at least 6.0 by semen (pH 7.2–8.2) to promote sperm survival. These observations provided the rationale for developing acid-buffering products as candidate multi-purpose agents that could serve as topical contraceptives and provide protection against acid-sensitive microbes. Two acid-buffering products, BufferGel® (developed by ReProtect Limited Liability Company, Baltimore, MD) and Acidform (developed by the Program for Topical Prevention of Conception and Disease at Rush University, Chicago, IL) were formulated as vaginal gels. BufferGel® was safe and well tolerated in women [8]–[10] and reduced the prevalence of bacterial vaginosis (BV) when applied twice daily for 2 weeks [9]. However, it did not alter the risk of HIV infection in a large-scale effectiveness trial [10]. When combined with a diaphragm, BufferGel® was as effective as N-9 for contraception; the 6-month pregnancy rate per hundred women was 10.1% (95% confidence interval [CI] 7.1–13.1%) for BufferGel® and 12.3 (95% CI 7.7–16.9) for N-9 spermicide users [11]. However, no reduction in pregnancy rate was observed when BufferGel® was used alone and dosed pericoitally [10]. 10.1371/journal.pone.0046901.g001Figure 1Trial profile. Acidform is a bioadhesive formulation that contains lactic acid as a primary buffering agent. In contrast, the active ingredient in BufferGel® is the hydrogen ion, which is released from the buffering agent Carbopol 974 [8]. Acidform buffers twice the volume of semen to maintain a pH of 4.45 in vitro, is spermicidal [7] and is active against herpes simplex virus (HSV), Chlamydia trachomatis and Neisseria gonorrhoeae in animal studies [12]–[14]. A randomized, blinded, crossover study was conducted among 20 sexually active sterilized women to compare the spermicidal effect of Acidform to that of a commercial 2% N-9 product. Acidform or N-9 product administered 0–30 min precoitus or Acidform given 8–10 h precoitus significantly reduced the mean number of progressively motile sperm compared to control cycles (0.19, 0.07, 0.75 vs. 17.94, respectively, p<0.05, Wilcoxon signed-rank test) [15]. Acidform has been marketed as a personal lubricant (AmphoraTM gel; Evofem Inc., San Diego, CA), and is currently being evaluated for contraceptive efficacy in a Phase III trial (ClinicalTrials.gov Identifier: NCT01306331). Prior Phase I safety studies of Acidform gel alone or in combination with a diaphragm have been conducted. No symptoms or irritation were reported by 6 women who used Acidform gel once daily for 6 days [16]. In a second study, women were randomized to Acidform (n = 44) or K-Y Jelly (n = 28) and applied gel twice daily for 14 days [17]. Twenty-seven women in the Acidform group (61%) compared to 8 women in the K-Y Jelly group (29%) reported at least one symptom of genital irritation (odds ratio = 2.62, CI 1.30–5.31, p = 0.009). There was a trend towards more safety events in women who used Acidform with a diaphragm for six months compared to women who used a diaphragm with K-Y Jelly [18]. 10.1371/journal.pone.0046901.t001 Table 1 Demographic data of recipients of Acidform and HEC placebo gel. Acidform Gel(N = 17) Placebo Gel(N = 18) p value Age in years (mean ± standard deviation) 30.15±7.17 32.16±9.42 0.48 Race (number, %) * 0.34 Black 6 (37.5%) 11 (61%) White 4 (25%) 3 (16.7%) Asian 0 1 (5.6%) Mixed 6 (37.5%) 3 (16.7%) Ethnicity 0.72 Hispanic 6 (35.3%) 5 (27.8%) Non-Hispanic 11 (64.7%) 13 (72.2%) Level of Education 0.64 Less than high school 0 1 (5.6) High school/General education diploma 5 (29.4%) 3 (16.7%) Some college 4 (23.5%) 6 (33.3%) College 6 (35.3%) 4 (22.2%) Graduate/Professional degree 2 (11.8%) 4 (22.2%) Number lifetime sex partners (median, range) 5 (1–34) 6 (0–100) 0.36 Reported history of anal sex 7 (41.2%) 7 (38.9%) 0.89 Current cigarette smoker 4 (23.5%) 4 (22.2%) 1.0 Tampon use 10 (58.8%) 12 (66.7%) 0.73 History of douching 5 (29.4%) 6 (33.3%) 1.0 Mean duration of menstrual cycle (days ± standard deviation) 28.6±2.12 28.1±1.76 0.42 Mean duration of menses (days ± standard deviation) 4.5±0.8 4.4±0.98 0.78 Current contraceptive method (number, %) Male condoms 7 (41.2%) 9 (50%) 0.74 Female condoms 1 (5.9%) 2 (11.1%) 1.0 Tubal ligation 1 (5.9%) 0 0.49 Intrauterine device 0 2 (11.1%) 0.49 Withdrawal 0 2 (11.1%) 0.49 Prior history of vaginitis (number, %) Candida vaginitis 10 (58.8%) 13 (72.2%) 0.49 Bacterial vaginosis 2 (11.8%) 6 (33.3%) 0.23 Prior history of STI (number, %) Trichomonas 0 2 (11.1%) 0.49 Chlamydia 3 (17.6%) 3 (16.7%) 1.0 Gonorrhea 1 (5.9%) 0 0.49 Genital warts 1 (5.9%) 0 0.49 HSV seropositivity (number, %) HSV-1 seropositive 13 (76.5%) 11 (61.1%) 0.47 HSV-2 seropositive 3 (17.6%) 5 (27.8%) 0.47 * For one participant in the Acidform gel group, race is unknown. The goal of the present study was to expand the safety assessment of Acidform gel with twice daily vaginal dosing over 14 days. Outcomes included symptoms, pelvic exam findings, concentrations of genital tract immune mediators, and quantification of antimicrobial activity of cervicovaginal secretions. BODY.METHODS.ETHICS STATEMENT: The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1. The study was conducted according to the Declaration of Helsinki and was approved by the Albert Einstein College of Medicine Institutional Review Board (IRB) and the NIAID Division of AIDS Prevention Science Review Committee. All study participants provided written informed consent. 10.1371/journal.pone.0046901.t002 Table 2 Adverse events related to Acidform and HEC placebo gel. Participant Number Gel Received Adverse Event Number of Episodes Duration 1 Acidform Vaginal Burning 3 1 min 4 Acidform Vaginal Burning 2 5 min, 20 min 5 Acidform Vulvar Itching 12 20 min 5 Acidform Vulvar Itching 5 5 min 5 Acidform Vulvar Erythema 1 3 days 9 Acidform Abdominal Cramping 1 15 min 11 Acidform Vulvar Dryness 1 2 days 13 Acidform Vulvar Burning 3 30 min 13 Acidform Vulvar Burning 1 10 min 13 Acidform Vulvar Itching 1 30 min 13 Acidform Vulvar Erythema 1 6 days 13 Acidform Vulvar Abrasion 1 3 days 16 Acidform Abdominal Cramping 1 60 min 16 Acidform Vaginal Bleeding 1 13 days 17 Acidform Abdominal Cramping 1 6.5 hours 18 Acidform Vaginitis 1 4 days 22 Acidform Vulvar Itching 2 10 min, 5 min 26 Acidform Vulvar Itching 1 15 min 3 Placebo Vulvar Itching 1 10 min 14 Placebo Vaginal Itching 2 5 min BODY.METHODS.PARTICIPANTS: Thirty-five healthy women between the ages of 18 and 50 years were recruited from the New York metropolitan area between February 2009 and December 2010. Inclusion criteria included regular menstrual cycles and willingness to abstain from sex for the duration of the study. Participants were excluded for pregnancy, breastfeeding, menopause, HIV infection, reproductive or urinary tract infection, bacterial vaginosis (BV), intermenstrual bleeding, abnormal Pap test, use of hormonal contraception during the study or in the previous two months, and antibiotic use in the week prior to enrollment. 10.1371/journal.pone.0046901.g002Figure 2The pH of the vagina and cervix was significantly lower 2 hours after application of Acidform compared to HEC placebo gel.Box-and-whisker plots showing the pH of the posterior fornix (a), lateral vaginal wall (b), cervix (c) and CVL (d) obtained at screening (Scr), 2 hours (2 h) and at Days 7, 14 and 21 after insertion of Acidform (white) or HEC placebo gel (gray). The line indicates the median values and the circles are outliers. The asterisks denote a significant difference between the Acidform and HEC placebo group. At screening, participants had urine collected for microscopy, culture and a pregnancy test. A gynecological examination was performed for detection of BV (wet preparation with Amsel clinical criteria), Trichomonas vaginalis (wet preparation), Candida species (KOH prep), and semen using an antibody immunoassay that detects p30, a glycoprotein produced by the prostate (Abacus Diagnostics, West Hills, CA). The pH was measured using a stainless steel sensor pH probe placed at the lateral vaginal wall, posterior fornix and cervix (ISFET PH77 Probe Hach Company, Loveland, CO). CVL was performed by washing the cervix and posterior fornix with 10 mL of normal saline (pH∼5.0). A Pap test was collected, and the presence of Neisseria gonorrhoeae and Chlamydia trachomatis infection was determined by nucleic acid amplification testing of endocervical swabs (Gen-Probe, Inc., San Diego, CA). Blood was collected for HIV ELISA, syphilis (rapid plasma reagin test), pregnancy, and serotype specific antibodies for HSV-1 and HSV-2 (HerpeSelect, Focus Diagnostics, Cypress, CA). The enrollment visit (Day 0) was completed within 45 days of screening and 2–6 days after cessation of menstrual bleeding to allow ample time for dosing prior to anticipated onset of subsequent menses. Tests for bacterial vaginosis, T. vaginalis, Candida species, pH and semen detection were repeated. A swab of the lateral vaginal wall was collected to assess changes in vaginal bacterial populations. Eligible participants were then randomized 1∶1 to receive Acidform or hydroxyethylcellulose (HEC) gel; the randomization was computer generated by the pharmacist. The gels differed in color, which precluded randomization in a double-blind fashion. However, study participants and laboratory personnel were not informed of the random assignments. The first dose of Acidform or HEC was administered intravaginally by a study clinician. Two hours post-insertion, the pH was measured and CVL was performed. Participants were instructed to apply a dose twice daily, preferably in the morning and at bedtime and to not insert gel when study visits were scheduled until after the visit. The participants were provided with a diary to record usage and symptoms. 10.1371/journal.pone.0046901.g003Figure 3The bactericidal activity of genital tract secretions against E. coli was significantly greater 2 hours after application of Acidform compared to HEC placebo gel.Box-and-whisker plots showing the percent inhibition of E. coli (a) and HSV-2 plaque formation (b) in CVL samples collected at screening (Scr), 2 hours (2 h) and at Days 7, 14 and 21 after insertion of Acidform (white) or HEC placebo gel (gray). The line indicates the median values and the circles are outliers. The asterisk denotes a significant difference between the Acidform and HEC placebo group. Subsequent study visits were conducted on Days 7 (range 5–9), 14 (range 14–16) and 21 (range 17–21). A speculum exam, wet mount microscopy, semen test, pH measurement and CVL were performed at each visit. At every visit, used and unused applicators were counted. Adverse event (AE) data were collected at each study visit and graded according to the NIH Division of AIDS Table for Grading the Severity of Adverse Events [19]. A swab of the lateral vaginal wall was collected on Day 14. BODY.METHODS.STUDY DRUGS: Acidform is a viscous, off-white gel containing three acidic compounds (lactic acid, citric acid and potassium bitartrate), a preservative (benzoic acid), two polymer thickeners (alginic acid and xantham gum), a humectant (glycerin), sodium hydroxide and water (pH 3.55). The placebo for this study is HEC gel, which is a water-based formulation that contains HEC, sodium chloride, sorbic acid and sodium hydroxide (pH 4.4) [20]. Both Acidform and HEC are applied with a single high-density polyethylene (HDPE) applicator capable of administering a 5 g (equal to 5 mL) dose of gel. Evofem, Inc. (formerly Instead Healthcare, LLC San Diego, CA) provided Acidform lubricant, HEC gel and HDPE applicators for this study. BODY.METHODS.CVL SAMPLES: CVL specimens were transported to the laboratory on ice and were clarified by centrifugation at 700 g for 10 minutes at 4°C. Supernatants were divided into aliquots and stored at −80°C. The protein concentration (Pierce Micro BCA) and pH (ColorpHast, pH 2–9, EMD Chemicals) of CVL samples were determined. 10.1371/journal.pone.0046901.t003 Table 3 Summary of concentrations of immune mediators and comparison of changes between the Acidform and HEC placebo groups. Immune Mediators Geometric Mean for Screening Geometric Mean for2 hours Geometric Meanfor Day 7 Geometric Meanfor Day 14 Geometric Meanfor Day 21 Estimated MeanDrug Effect P-value time effect a P-value drug effect Total Protein (µg/ml) 280.2 165.3 164.7 189.7 251.3 −12.3 <0.01 0.62 Lysozyme (ng/ml) b 251.8 89.8 265.6 182.1 348.0 −30.5 <0.01 0.74 Lactoferrin (ng/ml) 1125.52 289.5 567.8 518.4 1131.3 −603.9 <0.01 0.04 HNP 1–3 (pg/ml) c 34637.8 14816.6 19285.3 31984.3 65336.1 −3354.0 <0.01 0.82 SLPI (pg/ml) d 219915.8 43761.1 88929.6 84897.5 88627.7 −36836.8 <0.01 0.21 IgG (ng/ml) 4929.0 3282.0 3490.5 3741.7 8950.8 −3188.8 <0.01 0.06 IgA (ng/ml) 981.4 256.9 407.3 603.3 1279.9 −363.8 <0.01 0.05 IL-1α (pg/ml) 58.6 7.7 26.0 31.3 55.4 −10.6 <0.01 0.35 IL-1ra (pg/ml) 6934.1 1825.1 3908.5 5201.0 9247.4 −3845.3 <0.01 <0.01 IL-1β (pg/ml) 4.2 1.1 3.6 2.8 17.8 −1.1 <0.01 0.60 IL-6 (pg/ml) 7.3 4.3 3.2 5.2 17.3 −3.7 <0.01 0.13 IL-8 (pg/ml) 482.7 90.6 169.9 183.5 883.9 −202.0 <0.01 0.08 IFN-γ (pg/ml) 1.5 0.5 1.0 0.9 1.4 −0.3 <0.01 0.28 MIP-1α (pg/ml) LLOD: n = 9 n = 12 n = 8 n = 6 n = 2 n/a <0.01 0.64 <Q3: n = 17 n = 20 n = 22 n = 22 n = 19 ≥Q3: n = 9 n = 2 n = 4 n = 7 n = 12 MIP-1β (pg/ml) LLOD: n = 13 n = 13 n = 13 n = 9 n = 4 n/a <0.01 0.15 <Q3: n = 16 n = 18 n = 18 n = 19 n = 18 ≥Q3: n = 6 n = 3 n = 3 n = 7 n = 11 RANTES (pg/ml) LLOD: n = 9 n = 15 n = 16 n = 15 n = 4 n/a <0.01 0.55 <Q3: n = 21 n = 15 n = 16 n = 12 n = 20 ≥Q3: n = 5 n = 4 n = 2 n = 8 n = 9 Abbreviations: LLOD, lower limit of detection; Q3, 3 rd quartile; n/a, not applicable. a As there was no significant interaction between treatment group and time, p-values <0.05 indicate that immune mediators changed over time, independent of whether participants applied Acidform or HEC placebo gel. b Two values were assigned the highest standard multiplied by the dilution. c Three values were assigned the highest standard multiplied by the dilution. d One value was assigned the highest standard multiplied by the dilution. BODY.METHODS.ANTIMICROBIAL ACTIVITY OF CVL: The antimicrobial activity against HSV-2 and E. coli in CVL was measured as previously described [21]. For anti-HSV activity, Vero cells were infected with ∼50–200 pfu of HSV-2(G) mixed 1∶1 with each CVL or control buffer and plaques were counted after 48 hours. All samples were tested in duplicate in two independent experiments. To assess the bactericidal activity, E. coli (ATCC strain 4382627) was grown overnight to stationary phase and then 3 μl of bacteria (∼109 cfu/ml) were mixed with 27 μl of CVL or control genital tract buffer (20 mmol/L potassium phosphate, 60 mmol/L sodium chloride, 0.2 mg/ml albumin, pH 4.5) and incubated at 37°C for two hours. The mixtures were further diluted in buffer (to yield 800–1000 colonies on control plates) and plated on agar enriched with trypticase soy broth. Colonies were counted using ImageQuant TL v2005 after an overnight incubation at 37°C. All samples were tested in duplicate and the percent inhibition was calculated relative to control wells. BODY.METHODS.MEASUREMENT OF IMMUNE MEDIATORS: Interleukin (IL)-1α, IL-1β, IL-6, IL-8, interferon (IFN)-γ, IFNa2, IL-1ra (IL-1 receptor antagonist), macrophage inflammatory protein (MIP)-1α, MIP-1β, and regulated upon activation, normal T-cell expressed and secreted (RANTES) were quantified in each CVL sample using a multiplex proteome array with beads from Chemicon International (Billerica, MA), measured using Luminex100 (Austin, TX) and analyzed using StarStation (Applied Cytometry Systems, Sacramento, CA). The levels of all other mediators were determined using commercial ELISA kits: secretory leukocyte protease inhibitor (SLPI) (R & D Systems), lactoferrin (Calbiochem, San Diego, CA), human neutrophil peptides 1–3 (HNP1–3) (HyCult Biotechnology, Uden, The Netherlands), IgG and IgA (CygnusTechnologies, Southport, NC) and lysozyme (Alpco Diagnostics, Salem, NH). The lower limit of detection (LLOD) for each assay in pg/ml was: HNP1–3, 156; SLPI, 25; IgG, 100; IgA, 150; lactoferrin, 1000; lysozyme 500; IL-1α, 3.5; IL-1β, 0.4; IL-6, 0.3; IL-8, 0.2; IFN-γ, 0.1; IFN-α2, 24.5; IL-1ra, 2.9; MIP-1α, 3.5; MIP-1β, 4.5; RANTES, 1.0. Sample values that were below the level of the lowest standard were set at the midpoint between zero and the LLOD and then multiplied by the dilution factor. Concentrations that were above the highest detection concentration were repeated at higher dilutions or, if insufficient sample was available, were assigned the value of the highest standard multiplied by the dilution. BODY.METHODS.DYE STAIN ASSAY (DSA) OF APPLICATORS TO ASSESS ADHERENCE: Each participant received 30 pre-filled individually packaged applicators of Acidform or HEC gel and was asked to return used and unused applicators at each study visit. Drug was dispensed from the returned unused applicators ex vivo, and all the applicators were then batched and stained with 0.05% FD&C Blue #1 granular food dye (Prime Ingredients INC, Saddlebrook, NJ) to detect whether the applicators had been inserted vaginally [22], [23]. Applicators inserted by study staff and unused applicators dispensed ex vivo by staff were included as positive and negative controls, respectively. Two independent observers scored the applicators as exposed or unexposed to vaginal mucus, and results were compared to subjects' self-reports. BODY.METHODS.QUANTIFICATION OF VAGINAL MICROBIOTA: DNA was extracted from stored vaginal swabs as previously described, with one extraction control for every 12 swabs. [24]. Human 18S rRNA gene polymerase chain reaction (PCR) was performed on all extracted DNA samples to ensure contact with vaginal mucosa during sampling and the presence of amplifiable DNA; amplification control PCR targeting a jellyfish aequorin gene was used to exclude the presence of PCR inhibitors [25]. Quantitative PCR (qPCR) assays utilizing primers and probes specific to each bacterium's 16S rRNA gene were then used to quantify concentrations of key vaginal bacteria associated with health (Lactobacillus crispatus and Lactobacillus jensenii) and BV (Gardnerella vaginalis, Megasphaera-like bacterium (type 1 & type 2), and Clostridia-like bacterial vaginosis associated bacterium 2 (BVAB2)) [26]. Quantified bacterial levels were expressed as copies of bacterial DNA per vaginal swab. BODY.METHODS.STUDY OUTCOME AND STATISTICAL ANALYSIS: The primary objectives of this study were to examine the effect of Acidform or HEC placebo gel on antimicrobial activity and mediators of mucosal immunity. Secondary objectives were to assess the extent of acid buffering by Acidform after vaginal application and to evaluate a candidate biomarker of adherence. An exploratory objective was to evaluate changes in vaginal microbiota. Demographic data at baseline between the two groups were compared using chi-squared or Fisher's exact tests for categorical variables and using t-tests or Wilcoxon rank-sum tests for continuous variables. Differences in the rate of women with at least 1 AE between the two treatment groups were compared using Fisher's exact test. Data on immune mediators were log transformed, where appropriate. Linear mixed models with a random intercept were used to examine changes in immune mediators during the study period and the effects of treatment on changes in immune mediators. For 3 immune mediators, (MIP-1α, MIP-1β, and RANTES), where more than 20% of immune mediators were below the limit of detection, immune mediators were converted to 3-level variables: 0 if immune mediators were undetectable, 1 if immune mediators were detectable but less than the 3rd quartile of those with quantifiable levels, and 2 if immune mediators were at or above the 3rd quartile. Generalized linear mixed models with a random intercept and a cumulative logit link function were used to examine changes in these 3 immune mediators during the study period. An interaction between treatment and time was included in the model to examine whether changes in immune mediators varied with treatment. Wilcoxon rank-sum tests were used to compare differences in pH, antimicrobial activity, and vaginal microbiota between the two treatment groups. Differences in L. crispatus, L. jensenii, G. vaginalis measured at enrollment and 14 days after gel use were compared using Wilcoxon signed-rank tests. Bonferroni adjustments were applied for post hoc comparisons between visits. Spearman's correlation coefficients (SCC) were estimated to assess associations between antimicrobial activity and concentrations of immune mediators from screening samples. Agreement between two observers for differentiating applicators that were intravaginally inserted from those not intravaginally inserted was assessed using the kappa statistic. A kappa > = 0.75 indicated excellent agreement between the two observers. A kappa between 0.4 and 0.75 indicated fair to good agreement [27]. All statistical analyses were performed using SAS Version 9.2 (SAS Inc., Cary, NC, USA). All P values were two-tailed, with P<0.05 considered as statistically significant results. 10.1371/journal.pone.0046901.t004 Table 4 qPCR levels of bacteria from swabs collected at baseline (Day 0) and after 14 days of twice daily application of Acidform or HEC gel. Gel group Bacterium # PCR + baseline % PCR + baseline Median DNA copies/swabat baseline # PCR + Day 14 % PCR + Day 14 Median DNA copies/swab a at Day 14 Acidform (n = 17) L. crispatus 10 59 9.3×10 7 12 71 5.9×10 6 L. jensenii 11 65 7.6×10 6 12 71 1.4×10 7 G. vaginalis 10 59 1.3×10 6 8 47 3.6×10 4 Megasphaera 1 6 4.6×10 7* 1 6 1.8×10 7b BVAB2 1 6 1.8×10 7* 1 6 2.1×10 7b HEC (n = 18) L. crispatus 8 44 5.9×10 7 9 50 4.2×10 7 L. jensenii 9 50 6.8×10 6 9 50 1.1×10 7 G. vaginalis 16 89 9.8×10 5 13 72 4.4×10 6 Megasphaera 4 22 3.2×10 7 3 17 1.4×10 6 BVAB2 4 22 1.2×10 6 4 22 1.9×10 5 a Median DNA copies/swab among participants with detectable DNA. b DNA copies/swab for one participant. BODY.METHODS.SAMPLE SIZE: A total sample size of 36 (18 subjects in each arm) was selected a priori to allow 80% power to observe statistically significant equivalence of means if we assume that Acidform and HEC have no impact on anti-HSV activity and consider the mean difference of 1 standard deviation as the equivalence limit. BODY.RESULTS.STUDY SUBJECTS: Fifty-five women were assessed for eligibility, and 35 were enrolled (Fig. 1). Study product expired prior to enrollment of the final participant. The majority of exclusions were due to an abnormal Pap test (n = 8) or reproductive tract infection (n = 5). All 35 participants completed the trial, which included 17 in the Acidform and 18 in the placebo group. The mean age of the enrolled women was 31.2 years and there were no differences in race, ethnicity, education or other demographic characteristics between participants in the Acidform compared to the placebo group (Table 1). BODY.RESULTS.TOLERANCE OF ACIDFORM GEL: There were 51 AEs among 18 women. Forty-two of the AEs, which occurred among 13 women (11 in the Acidform and 2 in the HEC group), were possibly or probably related to the study products (Table 2). Eleven of 17 (65%) women who received Acidform had at least 1 AE compared with 2 of 18 (11%) who received HEC placebo gel (p = 0.002). The most commonly reported AEs were genital tract itching and burning. Two subjects developed vulvar erythema that was temporally linked to Acidform application. All product related AEs were graded as mild and most occurred immediately following gel application. BODY.RESULTS.EFFECTS OF ACIDFORM ON PH AND ON ANTIMICROBIAL ACTIVITY OF GENITAL SECRETIONS: The median pH at the cervix, lateral vaginal wall, posterior fornix and CVL was significantly lower 2 hours after application of Acidform compared to the placebo group (p<0.01) (Fig. 2). There were no significant differences between the groups in pH at any location at screening, Day 7, 14 or 21, although there was a trend towards lower pH at all locations on days 7 and 14 in the Acidform group. The activity of female genital secretions collected by CVL against E. coli and HSV was measured ex vivo at each time-point. The median percent inhibition of E. coli was 72 [interquartile range (IQR) 36–99] and 61 [42–95] at screening in the Acidform and HEC groups, respectively. Consistent with a lower pH 2 hours after Acidform gel application, we found that the bactericidal activity of CVL against E. coli, which are susceptible to lactic acid, was significantly greater and less variable 2 hours after initial application of Acidform compared to placebo (98 [96.5–99] for Acidform vs. 47 [33–65] for HEC, p<0.01, Fig. 3). However, bactericidal activity did not differ significantly between the drug and placebo group at any of the other time-points, which is consistent with the absence of any significant differences in pH at the other time-points (Figs. 2 and 3). The median percent inhibition of HSV-2 plaque formation was 76 [26–100] and 59 [21.5–10] at screening in the Acidform and HEC arms, respectively. There were no statistically significant differences in anti-HSV activity between the Acidform and placebo group after initial or repeated gel application (Fig. 3). BODY.RESULTS.EFFECTS OF ACIDFORM ON INFLAMMATORY MEDIATORS AND HOST PROTECTIVE FACTORS: The concentrations of the majority of cytokines, chemokines, and antimicrobial proteins decreased 7 and 14 days after vaginal gel use, independent of whether participants applied Acidform or HEC placebo gel, because there was no significant interaction between treatment group and time (Table 3). These time-points corresponded with menstrual cycle days 13–18 and 20–25, respectively, and likely reflect the physiological nadir [28]–[30]. There was a statistically significant drug effect observed for lactoferrin (p = 0.04) and IL-1ra (p<0.01) (Table 3). In the Acidform group, lactoferrin and IL-1ra concentrations in CVL were significantly lower compared to the HEC group. The estimated mean drug effects for lactoferrin and IL-1ra were −603.9 ng/ml and -3845.3 pg/ml, respectively (Table 3). No significant drug effect was observed for the other cytokines, chemokines and host protective factors. However, there was a trend towards a reduction in CVL concentrations of IgG (p = 0.06), IgA (p = 0.05) and IL-8 (p = 0.08) in women who applied Acidform gel. BODY.RESULTS.CORRELATION OF ANTIMICROBIAL ACTIVITY WITH MUCOSAL IMMUNE MEDIATORS AND VAGINAL PH: The E. coli bactericidal activity correlated negatively with the pH of the vaginal wall (ρ = -0.42, p = 0.01) and positively with total protein (ρ = 0.67, p<0.0001), but not other immune mediators. In contrast, the anti-HSV activity correlated modestly and significantly with concentrations of IL-1α (r = 0.55, p = 0.0005), IL-1β (r = 0.45, p = 0.007), IL-8 (r = 0.52, p = 0.001), HNP1–3 (r = 0.42, p = 0.01), lysozyme (r = 0.36, p = 0.03), and IgA (r = 0.34, p = 0.04), but not with vaginal pH (r = −0.11, p = 0.54). These correlations are consistent with results obtained in other studies [21], [31], [32]. BODY.RESULTS.EFFECTS OF ACIDFORM ON VAGINAL MICROBIOTA: The number of women with L. crispatus or L. jensenii detected at enrollment did not differ significantly between the two groups and represented 51% and 57%, respectively. There were no significant changes in the number of women with detectable L. crispatus or L. jensenii or in the concentrations of bacteria recovered by PCR in the Acidform or HEC group following 14 days of twice daily gel use (Table 4). However, there was a trend towards a decrease in the concentration of G. vaginalis following repeated application of Acidform from a median of 1.3×106 to 3.6×104 DNA copies/swab (p = 0.083). There were no cases of BV diagnosed by Amsel criteria throughout the study period. Five women had high levels of either Megasphaera or BVAB2 detected at the enrollment visit and 14 days later, suggesting that these women may have had unrecognized BV not revealed by Amsel clinical criteria. BODY.RESULTS.MEASUREMENT OF ADHERENCE BY APPLICATOR STAINING: Subjects were instructed to return used and unused applicators. The returned applicators were stained within four months along with positive (applicators inserted by study staff) and negative (unused applicators that had been dispensed ex vivo) controls. A total of 1012 of 1050 (96%) polyethylene applicators were returned, including 935 that participants reported had been intra-vaginally applied and 77 unopened pre-filled applicators. Four participants reported missing 1 dose, 2 reported missing 3 doses and 2 reported missing 5 doses of gel. Both observers correctly identified 29 of the 30 applicators inserted by study staff as positive and scored 87% and 96%, respectively, of the returned applicators that were reported to have been used as positive. The two observers identified 40/60 (66%) and 58/60 (96%) of the negative controls as negative and 55/77 (71%) and 58/77 (75%), respectively, of the returned unused applicators as negative. There was good agreement between the observers, as demonstrated by a kappa of 0.64, 95% confidence interval (0.58, 0.69). The sensitivity of the DSA for the two observers was 87% and 84%, respectively and the specificity was 69% and 85%, respectively. The positive predictive value was 95% and 97%, respectively and the negative predictive value was 44% and 43%, respectively. BODY.DISCUSSION: Acidform was found to be more irritating than HEC placebo gel, with a greater proportion of mild genital symptoms. A similar increased rate of mild AEs was observed in an earlier study when Acidform was compared to K-Y Jelly [17]. Despite the findings of mild irritation associated with Acidform use, we observed no increase in pro-inflammatory cytokines or chemokines, although there was a significant decrease in the concentration of the anti-inflammatory protein, IL-1ra in CVL obtained from participants who applied Aciform compared to HEC gel. Whether the mild irritation and the decrease in IL-1ra observed in this study portend a risk for mucosal inflammation with more prolonged exposure to Acidform requires further study. The median pH of the cervix and vagina 2 hours after a clinician administered Acidform gel was 3.5–3.7, which was associated with increased E. coli bactericidal activity, and is low enough to completely immobilize spermatozoa if the pH is maintained following coitus. However, no significant differences in bactericidal activity or pH at the lateral vaginal wall, posterior fornix or cervix were observed at any other time-point, which suggests that the acid-buffering effects do not persist. The average time elapsed between gel dosing and CVL sampling for the Day 7 and 14 study visits was 13 and 16 hours, respectively. These findings suggest that Acidform may be effective as a topical contraceptive and may prevent vaginal E. coli colonization, which has been observed after sexual intercourse with and without a condom [33]. We recently demonstrated an inverse correlation between vaginal E. coli colonization and bactericidal activity of CVL, indicating that the ex vivo activity may translate to protection against colonization [34]. However, the transient nature of the responses observed in the current study suggests that the product would have to be applied shortly prior to sex to be effective. The need to apply gel shortly prior to intercourse, the brief duration of activity, and the potential to develop genital irritation may limit adherence, acceptability and efficacy of Acidform gel as a topical contraceptive. Despite potent antiviral activity in a mouse model [12], there was no significant increase in anti-HSV activity of CVL following Acidform gel use. These findings may reflect the pH of CVL (mean of 4 at 2 hours and >4.5 at all other visits), which was likely not sufficient to inhibit HSV. We previously demonstrated that HSV inactivation in vitro is rapid and substantial at pH 3.5, but less effective at pH of 4.5 [12]. The anti-HSV activity of CVL correlates with concentrations of several immune mediators including lactoferrin, IL-8 and IgA; each of these was lower following Acidform use and this may have also contributed to the absence of any increase in anti-HSV activity. Notably, lactoferrin, an anti-bacterial glycoprotein produced by epithelial cells and neutrophils has been shown to inhibit HSV in vitro [35]. While we observed no loss in the anti-HSV activity of CVL in this study, the lower levels of lactoferrin and other immune mediators following Acidform gel application suggest that more prolonged or frequent exposure could potentially interfere with mucosal defense. Further studies are needed to determine the clinical significance of these findings. The healthy human vaginal microbiome is dominated by Lactobacillus species at high concentrations, including L. crispatus and L. jensenii [36], which maintain acidic vaginal pH by producing lactic acid. BV occurs when these beneficial vaginal lactobacilli are replaced by overgrowth of commensal vaginal anaerobes [36], [37]. In a previous Phase I study, there was no significant increase in hydrogen peroxide-producing lactobacilli following repeated vaginal application of Acidform gel, as measured by semiquantitative vaginal cultures [17]. In the present study, qPCR was utilized to assess the impact of Acidform gel on absolute quantities of protective lactobacilli and BV-associated organisms. There was no increase in the concentrations of beneficial lactobacilli following use of Acidform gel. Notably, there was a decrease in the amount of G. vaginalis recovered following Acidform, but not HEC gel application, on Day 14. Gardnerella vaginalis is almost always present in the vaginas of women with BV, but is also found in 70% of women without BV [26]. These findings suggest that the product may provide at least some colonization resistance against G. vaginalis, although further studies with sexually active women are needed to determine the potential role Acidform may play in promoting a healthy microbiome. Future studies should include more comprehensive studies of the effects of Acidform on the vaginal microbiota. This is the first study to apply the DSA to polyethylene applicators used to insert gel more than once daily. The DSA has been previously studied with methylcellulose, HEC, Carraguard, and PRO 2000 gels inserted once daily from low-density polyethylene (LDPE) applicators, with sensitivity and specificity of >90% [22], [38], [39]. The sensitivity of the assay was decreased when studied with VivaGel gel applied twice daily from polypropylene applicators [23], [39]. The difference may have been due to gel remaining in the vagina from the previous insertion. Alternatively, the sensitivity and specificity of the DSA may vary with the type of applicator used (polyethylene vs. polypropylene). In a recent study of tenofovir gel applied once daily with polypropylene applicators [21], the sensitivity of the DSA was >90% but the specificity was <70%, suggesting that staining of polypropylene applicators may not be as effective as that of polyethylene. In the current study of twice daily dosing of Acidform or HEC from polyethylene applicators, the sensitivity of the DSA for two observers was 87% and 84%, respectively and the specificity was 69% and 85%, respectively. These results suggest that the DSA may not be an effective method to assess applicator use when gel is administered more than once daily, despite the type of applicator used. Important limitations of the DSA are the differences in technique used by different groups and the high degree of interobserver variability [21], [23], [39], indicating the need for standardization and training to reduce subjectivity. These findings also highlight the need for better markers of adherence. A recent study suggests that direct inspection of polypropylene applicators under ultraviolet light may provide a reliable assessment of adherence [40]. In summary, Acidform gel was associated with a decrease in pH and an increase in E. coli bactericidal activity 2 hours after gel application as well as a decrease in the concentration of G. vaginalis recovered on Day 14, suggesting that it may promote a healthier vaginal microbial environment. However, twice daily application of Acidform was associated with mild irritation and lower CVL levels of several immune mediators compared to HEC placebo gel. Determination of the clinical significance of these findings requires additional safety studies of this candidate non-hormonal contraceptive with sexually active populations and more prolonged product exposure. BODY.SUPPORTING INFORMATION: Checklist S1 CONSORT Checklist. (DOC)Click here for additional data file. Protocol S1 Trial Protocol. (DOC)Click here for additional data file.

TITLE: A 6-week, multicentre, randomized controlled clinical trial to evaluate the safety and efficacy of placeboxetine hydrochloride in the treatment of major depressive disorder in an Indian setting ABSTRACT.INTRODUCTION:: This paper describes a fictitious study of a fictitious drug. A companion paper in this issue of the Indian J Psychiatry critically examines this paper and provides author, reader, reviewer, and researcher perspectives on problems related to the design and conduct of a clinical trial; on issues related to the analysis of data; on how to write a research paper; and on how to critically read or review a journal article. Readers are invited to appraise this paper and then compare their assessment with that presented in the companion paper. ABSTRACT.BACKGROUND:: This study sought to compare the safety and efficacy of placeboxetine (PB) hydrochloride extended release capsules with sertraline hydrochloride in patients diagnosed with major depressive disorder in 15 general hospitals in south India. ABSTRACT.MATERIALS AND METHODS:: In a prospective, open-label, 15-center, randomized controlled clinical trial, consecutive outpatients diagnosed with major depressive disorder of at least moderate severity were randomized 1:1 to receive flexible doses of either PB or sertraline once each morning. Patients were evaluated every two weeks, until the study endpoint, using the Hamilton Rating Scale for Depression (HAM-D) and the Montgomery-Asberg Rating Scale (MADRS). Safety was determined through assessments of vital signs, adverse events, study discontinuation rates, hematological parameters, metabolic parameters, electrocardiography, and other measures. ABSTRACT.RESULTS:: Ten patients dropped out of the study from each treatment arm. There was a significant, marked improvement in HAM-D and MADRS scores in each group by the treatment endpoint. There was no significant difference between PB and sertraline groups on either HAM-D or MADRS at any visit. The response rate was 90% with PB and 92% with sertraline. The remission rate was 70% with PB and 75% with sertraline. All laboratory parameters were within normal limits in all patients. There were no serious adverse events. ABSTRACT.CONCLUSIONS:: Placeboxetine is as safe and effective as sertraline in Indian patients with major depressive disorder. BODY.INTRODUCTION: Depression is a common disorder with an estimated incidence of 15-20%. Depression is associated with considerable family and socioeconomic burden. Patients with depression often suffer medical and psychiatric comorbidity.[1,2] Although various psychological and somatic treatments are available to treat depression, response and remission rates are low in clinical trials as well as in real world settings, and treatment-emergent adverse events are common. [3–5]There is therefore a need for continued efforts in research and development to introduce newer treatments which may have a more favorable efficacy and adverse effect profile than the existing treatments. Placeboxetine (PB) hydrochloride is one such newer treatment, recently approved by several regulatory agencies in the North American and European continents. PB belongs to the serotonin-norepinephrine (SNRI) class of antidepressant drugs. Other than its potent inhibition of the serotonin and norepinephrine transporter proteins, PB has no action at over 60 molecular targets, including receptor sites and enzymes; thereby, it can be expected to have a favorable adverse effect profile with little to no risk of pharmacokinetic drug interactions.[6,7] Four Phase III randomized, placebo-controlled trials (RCTs) conducted in 12 countries in North America and Europe found PB (75-300 mg/day) safe and effective in patient with major depressive disorder.[8–11] There are no data on the safety and efficacy of PB in Indian patients. The present study therefore compared PB with sertraline, an approved antidepressant, in patients with major depressive disorder in south India. BODY.MATERIALS AND METHODS.STUDY DESIGN: The present study was a 6-week, multicentre, open-label, randomized controlled trial conducted in patients recruited from the departments of psychiatry in 15 general hospitals in south India. Approval for the study was obtained from the Drug Controller General of India as well as the ethics committees at each participating site. The trial was registered at the Clinical Trials Registry of India. Informed consent for participation in the study was obtained from each patient in writing. BODY.MATERIALS AND METHODS.SAMPLE: The sample comprised consecutive consenting male and female patients, aged 18-65 years, diagnosed with major depressive disorder that was at least 4 weeks in duration. All patients were free from psychotropic medication for at least two weeks prior to randomization, and all scored at least 18 (indicating at least moderate severity of illness) on the Hamilton Rating Scale for Depression (HAM-D). Patients were excluded if they had any other major Axis I psychiatric disorder, any medical or neurological disorder that could influence the diagnosis or treatment of depression, any condition other than depression that was not on stable treatment for at least the past one month, any condition that could pose a health risk during a clinical trial, and any clinically significant abnormality or disorder that was newly detected during the baseline assessments. Patients who were considered to be at risk of suicide were also excluded from participation in the study. BODY.MATERIALS AND METHODS.TREATMENT: Consenting patients who fulfilled the study selection criteria were randomized 1:1 to receive open-label treatment with either PB extended release capsules (starting dose, 75 mg/day) or sertraline tablets (starting dose, 50 mg/day). All medications were dosed in the morning. PB was obtained from Indigent Pharma, Bogasapura, India, and sertraline was purchased from commercial sources. The dose of medication was flexibly uptitrated, based on efficacy and tolerability, at the discretion of the treating clinician. Downtitration was also allowed if raised doses were not tolerated. Thus, treatment reflected real world practice. The dosing range was 75-300 mg/day for PB and 50-200 mg/day for sertraline. No other psychotropic medication was permitted during the study except for lorazepam at a maximum dose of 2 mg during a 24-h period to treat agitation or insomnia. At the end of the 6-week study, the trial medications were tapered and withdrawn. BODY.MATERIALS AND METHODS.ASSESSMENTS: Patients were assessed at baseline and, again, at 2-weekly intervals using the HAM-D, the Montgomery-Asberg Rating Scale (MADRS), and the Clinical Global Impression Scales for Severity and Improvement (CGI-S and CGI-I). Response was defined as 50% or greater improvement in HAM-D scores, and remission as a final HAM-D score of 10 or lower. All adverse events were recorded. Safety assessments included assessment of vital signs, hematological measures, metabolic parameters, liver function tests, renal function tests, thyroid function tests, and the electrocardiogram (EKG). BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: The primary outcome measure was the improvement in the HAM-D score after 6 weeks of treatment. Secondary outcomes were post-baseline changes in MADRS ratings as well as safety measures. Means were compared within groups using the paired t test and between groups using the independent sample t test. Frequencies were compared between groups using the Chi square test. Alpha for statistical significance was set at 0.05. BODY.RESULTS: A total of 209 patients were screened. Nine patients did not meet the study selection criteria; thus, 200 patients were recruited, with 100 patients randomized to each group. Of these, 10 dropped out of each treatment arm, leaving 90 completers in each group. Reasons for drop out included adverse events (n=12), inefficacy (n=6), and withdrawal of consent (n=2). The reason for drop out could not be ascertained in one patient. The sociodemographic and clinical description of the sample is presented in Table 1. The sample was relatively young and predominantly male. The groups were comparable at baseline on all sociodemographic and clinical variables. Table 1 Sociodemographic and clinical description of the sample * At the 6-week study endpoint, the mean (standard deviation) dose of PB was 250.8 (37.4) and that of sertraline was 110.5 (26.9). The mean HAM-D scores across the course of the study are presented in Figure 1. There were no significant differences in HAM-D ratings between groups either at baseline or at any assessment point afterwards. The primary outcome, improvement in HAM-D scores between baseline and endpoint, was significant for both PB (P<0.001) and sertraline (P<0.001) groups; in fact, there was statistically significant improvement in HAM-D scores at the first (2-week) follow up, itself (P<0.01 for each group), indicating robust, early improvement with treatment. Figure 1Mean HAM-D scores during the course of the study in placeboxetine and sertraline groups Similar results were obtained with the MADRS assessments [Figure 2]. There was no difference between groups at baseline, there was significant improvement in each group at the end of weeks 2 (P<0.01) and 6 (P<0.001), and there were no significant differences between groups at any follow up visit. Figure 2Mean MADRS scores during the course of the study in placeboxetine and sertraline groups The response rate was 90% with PB and 92% with sertraline. The remission rate was 70% with PB and 75% with sertraline. The two groups did not differ significantly on either measure [Table 2]. Table 2 Response and remission rates with placeboxetine and sertraline * Table 3 presents adverse events that were reported by at least 2% of the patients in either group during the course of the study. Both drugs were well tolerated. Most of the adverse events occurred early during the course of the study. All adverse events recorded were transient and mild to moderate in severity. PB was associated with significantly less nausea and headache than sertraline; otherwise, adverse events did not differ significantly between the two groups. There were no serious adverse events during the study. Table 3 Adverse events reported by at least 2% of the patients in either group * There were no significant changes in heart rate or blood pressure at any time point. There were no abnormalities identified in the EKG or in any of the hematological and biochemical parameters studied. Metabolic parameters are presented in Table 4. There were no differences in metabolic parameters between groups at either baseline or endpoint, nor between baseline and endpoint in either group. Table 4 Metabolic parameters at baseline and endpoint in placeboxetine and sertraline groups * BODY.DISCUSSION: In this 15-centre, 6-week, parallel group, randomized controlled trial conducted in Indian outpatients with major depressive disorder that was at least moderate in severity, PB was as effective as sertraline in attenuating depression rating scores on the HAM-D and MADRS. Statistically significant improvement in HAM-D and MADRS scores were evident at the end of 2 weeks of treatment, itself. The response (90%) and remission (70%) rates with PB were high, and were comparable with those achieved with sertraline (92% and 75%, respectively). PB was very well tolerated and was associated with significantly less nausea and headache than sertraline; in contrast, no adverse event occurred at a greater frequency with PB relative to sertraline. PB did not adversely affect heart rate, blood pressure, hematological measures, biochemical measures, metabolic measures, and the EKG. These data confirm in Indian patients the favorable results obtained for PB in the Phase III regulatory trials conducted in the North American and European continents. [8–11] The recommended dosing range of PB is 75-300 mg/day, as identified in the Phase III clinical trials.[8–11]It is noteworthy that, in this Indian study, the mean endpoint dose was 250 mg/day; that is, towards the higher end of the dosing range. Despite the administration of these higher doses, PB was tolerated as well as or better than sertraline. PB therefore appears to be a useful addition to the therapeutic armamentarium for the treatment of major depressive disorder in Indian patients. BODY.SUMMARY: This is the first RCT of PB in Indian patients with major depressive disorder. PB is effective and well tolerated, and is as safe and effective as sertraline in this regard.

TITLE: Learning to experience side effects after antidepressant intake – Results from a randomized, controlled, double-blind study ABSTRACT.BACKGROUND: Side effects play a key role in patients' failure to take antidepressants. There is evidence that verbal suggestions and informed consent elicit expectations that can in turn trigger the occurrence of side effects. Prior experience or learning mechanisms are also assumed to contribute to the development of side effects, although their role has not been thoroughly investigated. In this study, we examined whether an antidepressant's side effects can be learned via Pavlovian conditioning. ABSTRACT.METHODS: Participants (n = 39) were randomly allocated to one of two groups and were exposed to a classical conditioning procedure. During acquisition, 19 participants received amitriptyline and 20 participants received a placebo pill. Pills were taken for four nights together with a novel-tasting drink. After a washout phase, both groups received a placebo pill together with the novel-tasting drink (evocation). Side effects were assessed via the Generic Assessment of Side Effects Scale prior to acquisition (baseline), after acquisition, and after evocation. A score of antidepressant-specific side effects was calculated. ABSTRACT.RESULTS: Participants taking amitriptyline reported significantly more antidepressant-specific side effects after acquisition compared to both baseline and the placebo group. After evocation, participants who underwent the conditioning procedure with amitriptyline reported significantly more antidepressant-specific side effects than those who never received amitriptyline, even though both groups received a placebo. ABSTRACT.CONCLUSIONS: Our results indicate that antidepressant side effects can be learned using a conditioning paradigm and evoked via a placebo pill when applied with the same contextual factors as the verum. BODY.INTRODUCTION: The prescribing of antidepressants has risen over recent years, with up to 13.4 % of individuals in Western countries having been prescribed antidepressant medication at least once per year (Sihvo et al. 2010; Lockhart and Guthrie 2011; Mojtabai and Olfson 2014; Abbing-Karahagopian et al. 2014). Although antidepressants are effective in treating major depression (Cleare et al. 2015), patients often discontinue drug intake (Sawada et al. 2009; Bocquier et al. 2014). Rates of reported non-adherence vary, but some studies report rates of discontinuing antidepressant medication of over 50 % within the first 2 to 4 months (Serna et al. 2010), while others report even higher discontinuation rates (Bocquier et al. 2014). These rates are alarming, considering that guidelines suggest taking antidepressant medication for at least 6 to 9 months to prevent relapse after the remission of a depressive episode (Cleare et al. 2015). Several factors contributing to patients' non-adherence have been identified (Serna et al. 2010; De las Cuevas et al. 2014; Bocquier et al. 2014), but one particular factor emerges consistently as a reason for discontinuing antidepressants, namely, side effects (e.g., Serna et al. 2010; Hung et al. 2011; Murata and Kanbayashi 2012; De las Cuevas et al. 2014). Common side effects of antidepressants (i.e., pharmacological reactions due to drug intake that differ from those intended) are, for instance, daytime sleepiness, dry mouth, loss of interest in sexual activity, and weight gain (Ashton et al. 2005). Some of the adverse events occurring after medication intake can be attributed to the drug's specific pharmacological action, and many such events are considered to be dose dependent, whereas others, not attributable to the drug's pharmacological action, often appear to be dosage independent (Shedden Mora et al. 2011). The latter events can be studied in placebo groups in drug trials. All side effects occurring after the intake of an inert substance are not specific or attributable to the drugs' pharmacokinetics (Schedlowski et al. 2015). The occurrence of side effects after placebo intake is called the nocebo effect. Originally, it was assumed that some nocebo side effects occur due to the misattribution of pre-existing symptoms (Barsky et al. 2002). More recent studies have additionally shown that the adverse effects occurring in placebo groups in drug trials match the side effects reported in the active drug arms of these trials (e.g., Rief et al. 2009; Amanzio et al. 2009; Mitsikostas 2012). One explanation for the nocebo phenomenon is patients' expectations about possible side effects in general (Nestoriuc et al. 2010), which might be triggered by the information provided in the informed consent or by verbal suggestion (Mondaini et al. 2007; Cohen 2014). Another factor potentially contributing to the occurrence of side effects is prior experience or learning (Amanzio 2015). One such example is cancer patients experiencing nausea as a side effect after undergoing chemotherapy. It is assumed that initially, neutral stimuli such as the room in which the therapy is administered are associated with the occurrence of nausea; therefore, just entering the room can cause anticipatory nausea after a while (Matteson et al. 2002). Such conditioning effects can be generated if an originally neutral stimulus (NS) (e.g., the room) is combined with an active stimulus (unconditioned stimulus (UCS)) (e.g., chemotherapy) that leads to certain reactions (e.g., nausea). After several pairings of NS and UCS (acquisition phase), the NS becomes a conditioned stimulus (CS). This means that the CS alone can evoke the reaction that was originally generated by the UCS (evocation; Pavlov 2010). Although some authors differentiate between expectations and conditioning as different mechanisms involved in placebo and nocebo responses (Enck et al. 2013), it is not always possible to clearly distinguish them since learning also leads to certain expectations (Stewart-Williams and Podd 2004). Therefore, in this article, we do not differentiate between expectation and conditioning per se but rather between "expectation through verbal suggestion" and "learning/conditioning." Learning effects have been experimentally investigated, showing, for example, that with motion sickness, a nocebo response can be learned (Klosterhalfen et al. 2009). Colloca et al. (2008) found in a conditioning paradigm that a light paired with a noxious stimulus can induce a hyperalgesic nocebo effect in the evocation trial. In a subsequent study using a similar paradigm, they showed that even one acquisition trial suffices to induce nocebo effects, although effects are more stable after additional trials (Colloca et al. 2010). Conditioned nocebo effects can also be evoked by non-conscious stimuli (Jensen et al. 2012). When it comes to pharmacological responses, the role of conditioning has been demonstrated in conjunction with immune reactions (Albring et al. 2012). However, to the best of our knowledge, there has been no evidence forthcoming that reveals whether Pavlovian conditioning contributes to the development and maintenance of antidepressant side effects. We hypothesized that participants taking amitriptyline would report more antidepressant-specific side effects in all after four nights of medication intake (acquisition phase, i.e., pill intake combined with a novel-tasting drink as NS) and attribute more of these side effects to the medication intake than would participants taking a placebo. Furthermore, we hypothesized that after having undergone the aforementioned acquisition and a subsequent washout phase, receiving a placebo pill together with the novel-tasting drink (evocation) would lead to more reported antidepressant-specific side effects in total and more medication-attributed antidepressant-specific side effects in the group that had previously taken amitriptyline than in the placebo group. BODY.METHODS.PARTICIPANTS AND ETHICS: This study was conducted in the Division of Clinical Psychology at the Philipp University of Marburg in 2014. Participants aged between 18 and 69 years who were willing to refrain from alcohol consumption and driving during the study period were recruited via an advertisement at the university. To ascertain that only physically and mentally healthy participants were included, all subjects underwent a medical and psychological examination (by a study physician and a psychologist, both trained in Good Clinical Practice). These included interviews about medical history and mental health (according to the International Diagnosis Checklists; Hiller et al. 2004), an electrocardiogram, blood tests, and a urine pregnancy test (only in females). If the examinations yielded evidence of contraindications to the study medication as mentioned in the information sheet for health professionals, those participants were excluded. Prior to the beginning of the study, participants were informed about the study design and treatment by the study physician. Written informed consent was obtained from all individual participants included in the study. The experiment was conducted according to the Declaration of Helsinki. Since the current study was only an exploratory subinvestigation in addition to the main study (for detailed results, see Winkler et al. 2016), only the main study was registered at http://www.clinicaltrials.gov (NCT02127736). Nevertheless, the outcomes used in this study were determined as secondary outcome measures in the study protocol, which was approved by the ethics committee of the medical chamber of Hessen (Landesärztekammer Hessen; FF51/2013). Participants were paid for study participation. BODY.METHODS.EXPERIMENTAL DESIGN: After the medical and psychological examination, equal numbers of participants were randomized into the placebo and antidepressant groups; no stratification was conducted. Randomization was done by an independent researcher. Through randomization, each individual got a number, which was assigned to a medication container which held either placebo or antidepressant pills. Both experimenters and participants were blinded to group allocation. The experimental group received amitriptyline; the control group received identical-looking placebo pills. At the baseline assessment, all subjects filled in the Generic Assessment of Side Effects Scale (GASE; Rief et al. 2010). Afterwards, participants in the experimental group underwent a classical conditioning paradigm (see Fig. 1). During the acquisition phase (nights 1 to 4), participants received 50 mg of amitriptyline (US) together with 100 ml of a novel-tasting drink that consisted of lychee juice with woodruff syrup and blue food coloring. The drink was the neutral stimulus (NS), which was supposed to become the CS. The drinks' ingredients were chosen in order to increase the novelty, saliency, and distinctiveness of the CS, since it has been argued that this might increase the conditioned response (Doering and Rief 2012). Amitriptyline-neuraxpharm 50 mg was used and encapsulated for study purposes by licensed pharmacologists. To make pill intake more salient, the novel-tasting drink was used. Participants were instructed to take the medication and the novel-tasting drink immediately before going to bed on four subsequent nights. Once the acquisition phase was over, side effects during acquisition were assessed. The acquisition was followed by a 3-day washout phase (nights 5 to 7). On night 8, the evocation night, all participants received a placebo pill together with the novel-tasting drink (CS). The next day, side effects after evocation were assessed. The placebo control group underwent the same procedure as the experimental group but received placebo pills instead of amitriptyline during the acquisition phase.Fig. 1Experimental design BODY.METHODS.MEASURES: Side effects were assessed with the GASE (Rief et al. 2010). The GASE contains a list of 36 symptoms and covers the most frequently reported side effects in clinical trials using different drugs according to FDA. The patient gives a rating for the presence and severity of each of these symptoms on a 4-point Likert scale ranging from "not present" (0) to "severe" (3). In addition, the patient indicates for each symptom whether he or she thinks it is caused by the intake of the drug (yes/no). A total score can be calculated as a sum of all item answers (general symptom load) as well as a total score of only medication-attributed symptoms. The GASE reveals good internal consistency with Cronbach's α = 0.89 and has been validated in a large sample with more than 2500 participants (Rief et al. 2010). BODY.METHODS.MEASURES.PRIMARY OUTCOME MEASURE: For the purpose of our study, an Antidepressant Composite Score (GASE-AD) was calculated to assess side effects specific for the study's antidepressant. To assess the most frequently reported side effects, we chose items that at least 50 % of the experimental group had experienced after the acquisition phase. This criterion left us with four items: (1) dry mouth, (2) dizziness, (3) cardiovascular problems, and (4) fatigue or loss of energy. These symptoms are also listed in the Physician's Desk Reference (Barnhart 1988) and in the Compendium of Psychiatric Pharmacotherapy (Benkert and Hippius 2014) as common symptoms of amitriptyline. In addition, these 4 symptoms are listed among 12 very common symptoms of amitriptyline on http://www.pharmawiki.ch (2015). We then calculated the score of all reported antidepressant specific side effects (GASE-AD) and that of all medication-attributed antidepressant specific side effects (GASE-AD-MA). Detailed analyses of potentially positive placebo effects are reported elsewhere (Winkler et al. 2016). BODY.METHODS.MEASURES.FURTHER ANALYSES: In addition to studying antidepressant-specific side effects, we analyzed more generic side effects or symptoms also, since symptoms not specific to the drug under investigation can also occur after taking a placebo pill (Barsky et al. 2002). For this purpose, the four antidepressant-specific items were excluded from calculating the scales for all reported generic, i.e., not antidepressant-specific side effects (GASE-generic) and for all medication-attributed generic side effects (GASE-generic-MA). To give a complete overview of reported side effects, we also analyzed the complete GASE scale (GASE-total) and calculated a score for all common side effects of amitriptyline (GASE-AMI) independent of how often they were named in the experimental group after acquisition. This score contains the items that are mentioned as the most frequently reported side effects both in the Compendium of Psychiatric Pharmacotherapy (Benkert and Hippius 2014) and on http://www.pharmawiki.ch. The items in the score are (1) dry mouth, (2) dizziness, (3) cardiovascular problems, (4) fatigue or loss of energy, (5) palpitations or irregular heartbeat, (6) constipation, (7) abnormal sweating, and (8) tremor. Weight gain was not included in the score since participants only took amitriptyline for 4 days. In addition, accommodation problems were also not included in the score because it was not assessed in the GASE. For the GASE-total and the GASE-AMI, medication-attributed scores were calculated as well (GASE-total-MA and GASE-AMI-MA). To assess whether participants were unblinded by amitriptyline's experienced side effect profile, we asked the participants after the acquisition phase to guess which experimental group (amitriptyline vs. placebo) they belonged to. After study completion and unblinding, this rating (perceived group allocation) was correlated with current group allocation. To analyze any clinical correlates with the nocebo response, we applied the subscales of the Symptom Checklist-90-Revised (SCL-90-R; Derogatis 1994) and the Beck Depression Inventory (BDI; Beck et al. 1961), which were assessed at baseline and correlated those with the GASE-AD and GASE-AD-MA. BODY.METHODS.STATISTICAL ANALYSES: We calculated the sample size with G*power (Faul et al. 2007). The initial sample size for the study was calculated for the primary outcome in the main study (Winkler et al. 2016); hence, only 40 participants were recruited. However, for the current investigation, the sample size was calculated post hoc and revealed that in order to detect a large time × group interaction effect with a power of 80 % and an α level of 0.05, the estimated total sample size was n = 42. Statistical analyses were performed with IBM SPSS Statistics 21.0. Baseline characteristics were analyzed using t tests and χ 2 tests. Missing values in the GASE were replaced by multiple imputation. To test for differences in total side effect reporting and medication-attributed side effect reporting between and within groups, multivariate analyses of variance (MANOVA) for repeated measures with the factors time (baseline, acquisition, evocation) and group (amitriptyline or placebo) were applied. Significant effects in the MANOVA were followed up by pairwise comparisons. The pairwise comparisons were adjusted according to the Bonferroni's procedure; i.e., the within-group tests were adjusted for three comparisons each. The correlation between current group allocation and perceived group allocation was calculated via the phi coefficient. Correlations between the SCL-90-R subscales and the BDI and the GASE scales were calculated using the Pearson's correlation coefficient. BODY.RESULTS: Forty participants were recruited and randomized equally to the two groups. In the experimental group, one participant discontinued drug intake due to side effects and was therefore excluded from study participation. Nineteen subjects in the amitriptyline group and 20 subjects in the placebo control group were thus included in our analyses (see Fig. 2). There were no significant differences in age, sex, or weight between participants in the two groups at baseline (see Table 1).Fig. 2Flowchart Table 1Sample characteristicsCharacteristicsAmitriptyline (n = 19)Placebo (n = 20)Group differencesAge in years, M (SD)24.4 (3.5)23.6 (3.7) t (37) = −0.71, p = .481Number females, n (%)11 (57.9)11 (55.0) χ 2 (1) = 0.03, p = .556Weight in kg, M (SD)67.5 (11.3)63.9 (9.0) t (36a) = −1.09, p = .285 aOne participant in the placebo group did not answer this question BODY.RESULTS.PRIMARY OUTCOME—ANTIDEPRESSANT-SPECIFIC SIDE EFFECTS.GASE-AD (ANTIDEPRESSANT-SPECIFIC SIDE EFFECTS): Overall multivariate analyses offered the basis for subsequent pairwise comparisons of single conditions (group effect F (2, 36) = 13.26, p ≤ .001; time effect F (4, 34) = 10.33, p ≤ .001; group × time interaction effect F (4, 34) = 8.17, p ≤ .001; univariate analyses: group effect F (1, 37) = 11.27, p = .002; time effect F (1, 37) = 14.57, p ≤ .001; group × time interaction effect F (1, 37) = 14.37, p ≤ .001). We observed that the two groups differed significantly in reported antidepressant-specific side effects after the acquisition phase (p ≤ .001; effect size Hedge's g = 1.56; 95 % confidence interval (CI) 0.84–2.28) and after the evocation night (p = .045; g = 0.66; CI 0.01–1.30); the amitriptyline group reported significantly more side effects (see Table 2 and Fig. 3a). Furthermore, the amitriptyline group displayed significant differences between baseline and acquisition (p ≤ .001), between baseline and evocation (p = .007), and between acquisition and evocation (p ≤ .001). After the acquisition phase, subjects in the experimental group reported significantly more side effects compared with baseline and evocation. After the evocation night, participants also reported significantly more side effects compared with baseline.Table 2Means, standard deviations, and F-statistics for the univariate analyses for the different side effect scoresAmitriptyline M (SD)Placebo M (SD)Time effectGroup effectInteractionPrimary outcome GASE-AD F (1, 37) = 14.57** F (1, 37) = 11.27* F (1, 37) = 14.37** Baseline0.89 (0.88)1.10 (1.37) Acquisition4.37 (2.45)1.10 (1.59) Evocation2.35 (2.49)0.98 (1.51)GASE-AD-MA F (1, 37) = 17.31** F (1, 37) = 27.21** F (1, 37) = 14.12** Baseline0 (0)0 (0) Acquisition3.48 (2.59)0.18 (0.51) Evocation1.74 (2.58)0.10 (0.45)Further analysesGASE-generic F (1, 37) = 0.84 F (1, 37) = 0.23 F (1, 37) = 2.05 Baseline4.32 (3.68)3.75 (3.68) Acquisition5.63 (4.78)3.85 (3.69) Evocation3.68 (3.43)4.42 (5.49)GASE-generic-MA F (0.60, 22.20)a = 5.43* F (0.60, 22.20)a = 3.88 F (0.60, 22.20)a = 2.87 Baseline0 (0)0 (0) Acquisition1.87 (3.40)0.37 (1.09) Evocation0.48 (0.91)0.21 (0.90)GASE-total F (0.85, 31.48)a = 5.61* F (0.85, 31.48)a = 2.13 F (0.85, 31.48)a = 6.11* Baseline5.21 (3.55)4.85 (4.80) Acquisition10.00 (5.24)4.95 (4.95) Evocation6.03 (4.58)5.40 (6.77)GASE-total-MA F (0.76, 22.28)a = 14.53** F (0.76, 22.28)a = 23.45** F (0.76, 22.28)a = 10.04* Baseline0 (0)0 (0) Acquisition5.36 (4.69)0.55 (1.57) Evocation2.22 (3.17)0.31 (1.34)GASE-AMI F (1, 37) = 14.52** F (1, 37) = 9.13* F (1, 37) = 15.08** Baseline1.05 (1.13)1.40 (1.89) Acquisition5.33 (2.91)1.35 (2.11) Evocation2.87 (2.84)1.28 (2.41)GASE-AMI-MA F (1, 37) = 17.01** F (1, 37) = 31.54** F (1, 37) = 14.98** Baseline0 (0)0 (0) Acquisition4.06 (2.86)0.18 (0.51) Evocation1.89 (2.88)0.10 (0.45) GASE-AD Antidepressant Composite Score of the Generic Assessment of Side Effects Scale, GASE-AD-MA medication-attributed symptoms of the Antidepressant Composite Score of the Generic Assessment of Side Effects Scale, GASE-generic generic symptoms on the Generic Assessment of Side Effects Scale, GASE-generic-MA medication-attributed generic symptoms on the Generic Assessment of Side Effects Scale, GASE-total all reported side effects as assessed with the Generic Assessment of Side Effects Scale, GASE-total-MA all medication-attributed side effects as assessed with the Generic Assessment of Side Effects Scale, GASE-AMI score of all common side effects of amitriptyline, GASE-AMI-MA score of all medication-attributed common side effects of amitriptyline*p ≤ .05**p ≤ .001 aDegrees of freedom have been corrected according to Greenhous-Geisser Fig. 3Antidepressant-specific side effects for both groups and all time points. GASE-AD Antidepressant Composite Score of the Generic Assessment of Side Effects Scale, GASE-AD-MA medication-attributed symptoms of the Antidepressant Composite Score of the Generic Assessment of Side Effects Scale BODY.RESULTS.PRIMARY OUTCOME—ANTIDEPRESSANT-SPECIFIC SIDE EFFECTS.GASE-AD-MA (MEDICATION-ATTRIBUTED ANTIDEPRESSANT-SPECIFIC SIDE EFFECTS): We found that the amitriptyline group's medication-attributed, antidepressant-specific side effect score was significantly higher after the acquisition phase (p ≤ .001; g = 1.75; CI 1.02–2.49) and after the evocation night (p = .008; g = 0.88; CI 0.22–1.54) than the placebo group's (see Table 2 and Fig. 3b). We also noted significant within-group differences between both baseline and acquisition (p ≤ .001) and between baseline and evocation (p = .001) on the GASE-AD-MA in the amitriptyline group. Thus, more antidepressant-specific side effects were attributed to the medication after the acquisition phase (i.e., intake of amitriptyline for four nights) than at baseline. More importantly, however, more antidepressant-specific side effects were also reported as medication-attributed after evocation night (i.e., after placebo intake) than at baseline. Furthermore, the amitriptyline group also reported significantly more medication-attributed symptoms after the acquisition phase than after the evocation night (p = .006; univariate analyses: group effect F (1, 37) = 27.21, p ≤ .001; time effect F (1, 37) = 17.31, p ≤ .001; group × time interaction effect F (1, 37) = 14.12, p ≤ .001). BODY.RESULTS.FURTHER ANALYSES.GASE-GENERIC (GENERIC SIDE EFFECTS): Our results reveal that only in the amitriptyline group, there was a significant difference between baseline and acquisition phase in medication-attributed generic side effects (GASE-generic-MA; p = .007). Participants attributed more generic symptoms to the medication after the acquisition phase than at baseline. We observed no differences between the groups in either the GASE-generic or GASE-generic-MA (multivariate analyses: group effect F (2, 36) = 1.89, p = .166; time effect F (4, 34) = 2.83, p = .040; group × time interaction effect F (4, 34) = 2.19, p = .091; univariate analyses regarding GASE-generic-MA: time effect F (0.60, 22.20) = 5.43, p = .027). BODY.RESULTS.FURTHER ANALYSES.GASE-TOTAL (ALL SIDE EFFECTS): The experimental group reported significantly more total side effects (GASE-total) at acquisition than the control group (p = .003), and it reported significantly more medication-attributed total side effects (GASE-total-MA) at acquisition (p ≤ .001) and at evocation (p = .018). Only for the experimental group, significant within-group differences between the assessment points could be observed for the GASE-total score between baseline and acquisition (p ≤ .001) and between acquisition and evocation (p = .008). For the GASE-total-MA, significant differences between all assessment points could be observed in the experimental group (multivariate analyses: group effect F (2, 36) = 11.46, p ≤ .001; time effect F (4, 34) = 8.80, p ≤ .001; group × time interaction effect F (4, 34) = 6.75, p ≤ .001; for detailed results of the univariate analyses, see Table 2). BODY.RESULTS.FURTHER ANALYSES.GASE-AMI (COMMON SIDE EFFECTS OF AMITRIPTYLINE): Pairwise comparisons showed that there were significant group differences at acquisition in the GASE-AMI score (p ≤ .001) with the experimental group reporting more side effects. In the medication-attributed score for common side effects of amitriptyline (GASE-AMI-MA), groups differed significantly at acquisition (p ≤ .001) and at evocation (p = .009) in the direction that the experimental group reported more side effects. For the experimental group, within-group differences were significant for comparisons between all time points on the GASE-AMI and on the GASE-AMI-MA (multivariate analyses: group effect F (2, 36) = 15.35, p ≤ .001; time effect F (4, 34) = 11.61, p ≤ .001; group × time interaction effect F (4, 34) = 9.89, p ≤ .001; for detailed results of the univariate analyses, see Table 2). BODY.RESULTS.FURTHER ANALYSES.PERCEIVED GROUP ALLOCATION: Group allocation as rated subjectively by the participants after the acquisition phase (perceived group allocation) correlated significantly with actual group allocation (φ = .641), meaning that 82 % of participants guessed their group allocation correctly, indicating the participants' at least partial unblinding to group allocation. BODY.RESULTS.FURTHER ANALYSES.NOCEBO RESPONSE CORRELATES: We detected no significant correlations among either the SCL-90-R subscales or BDI and the GASE-AD and GASE-AD-MA at any time point (baseline, acquisition, or evocation) in the amitriptyline group, indicating the nocebo response's independence of these clinical features. BODY.DISCUSSION: The aim of our study was to investigate whether antidepressant-specific side effects are not only caused by the drug's pharmacological actions but also learned through classical conditioning. We found that antidepressant-specific side effects can be evoked by an identical-looking placebo pill in participants who had previously taken an antidepressant that was accompanied by the same stimulus (novel-tasting drink) as the intake of placebo. In addition, participants who had previously been taking the antidepressant rated more of the side effects after taking the placebo pill as being medication-induced than participants who had been taking the placebo all the time. In contrast to the antidepressants' specific side effects, the generic side effect score did not change significantly between the assessment points, although participants taking amitriptyline attributed more of these generic side effects to medication intake after acquisition. These findings suggest that learning plays a role in the experiencing and reporting of side effects from antidepressants. This result is highly relevant, since patients suffering from depression often experience several depressive episodes in their lives and usually undergo repeated pharmacological treatment (Solomon and Keller 2000). There is evidence that patients who have been prescribed antidepressant medication once are more likely to be prescribed antidepressants again (Sirey and Meyers 2014). Our results suggest that if a participant has had negative experiences with a certain drug before, learning processes may contribute to the re-occurrence of these side effects. This in turn may lead to non-adherence or drug discontinuation (e.g., Serna et al. 2010; Hung et al. 2011; Murata and Kanbayashi 2012; De las Cuevas et al. 2014) and hence to a worse outcome or higher risk of relapses (Åkerblad et al. 2006). Given that side effects seem to depend on prior experience, it would seem advisable to systematically assess a patient's prior experience with specific drugs before issuing a prescription and in case of negative experiences to try another drug (Doering and Rief 2013). Our finding that certain side effects can be learned is in line with research showing that learning plays an important role in nocebo effects (e.g., Colloca et al. 2008; Klosterhalfen et al. 2009). Furthermore, it also falls in line with studies demonstrating that pharmacological responses can be conditioned (Goebel et al. 2008; Attwood et al. 2010; Albring et al. 2012). There have been proposals and even tests involving conditioning procedures to reduce drug doses in pharmacotherapy, a mechanism called placebo-controlled dose reduction (Ader et al. 2010; for a review, see Doering and Rief 2012). The maintenance of a drug's therapeutic effect while possibly reducing side effects and hence enhancing compliance has been postulated as one advantage of placebo-controlled dose reduction (Doering and Rief 2012). However, both the placebo effect of drug intake can obviously be learned, as can the nocebo effect, a factor that should be considered when planning placebo-controlled dose reduction. Participants taking amitriptyline attributed more generic symptoms to medication intake, although their generic-symptom score after acquisition was not significantly higher than their own baseline and the placebo group's scores, revealing that part of the side effects patients report may be due to the misattribution of pre-existing symptoms. By thoroughly assessing symptoms and side effects including baseline evaluations (Rief et al. 2006), we have succeeded in demonstrating this "misassignment" of symptom attribution. This is an already-described phenomenon (Barsky et al. 2002). The effect in our study was admittedly rather small and needs to be replicated. However, it is an extremely relevant phenomenon in pharmacotherapy since the attribution of side effects to the medication is an important factor behind the discontinuation of medication intake. Some shortcomings of the present study should be mentioned. First, we only assessed subjective data as outcome measures. Participants may have reported more symptoms in general because they were taking a drug, even though they did not attribute them to that drug. To account for this bias, we differentiated between reported symptoms in general and medication-attributed symptoms. In addition, the structured assessment of side effects (rather than an unstructured evaluation) may trigger more reported side effects (Rief et al. 2006). Secondly, the correlation between perceived group allocation and actual group allocation indicates that at least some participants were unblinded to group allocation, a problem reported and discussed in previous antidepressant trials in general (Jeffrey et al. 1986; Margraf et al. 1991). In terms of our study, this might imply that the unblinding shaped the participants' expectations regarding the pill in the evocation night, with the amitriptyline group expecting more side effects. Thirdly, the generalizability of our results to a clinical setting is limited since we only examined healthy young individuals and always paired the drug intake with a salient new stimulus. Hence, we cannot conclude whether a paradigm in which only the pill's appearance (without a salient new stimulus) in the typical treatment context serves as the conditioned stimulus (the case in natural clinical settings) would evoke the same amount of side effects. A fourth limitation is that since this study was just a pilot trial, we did not incorporate an untreated group in the study design, something other researchers suggest (Colloca and Miller 2011). Finally, it is a shortcoming that it would have been advantageous to include an assessment of side effects after the washout phase in order to control for any residual symptoms from the acquisition phase. One could argue that the side effects occurring in the evocation night were only due to a residual concentration of amitriptyline in the blood. However, there is solid evidence that the plasma half-life of tricylic antidepressants ranges from 10 to 28 h (Rudorfer and Potter 1999). Our washout phase entailed three nights without medication intake, which is 86 h between the last intake of amitriptyline and intake of placebo and should rule out the argument that a residual concentration of amitriptyline might have accounted for the difference in the evocation night. In addition, our participants received only low doses of amitriptyline (50 mg). Nevertheless, one could also argue that the antidepressant-specific side effects reported in the evocation night were due to discontinuation effects of amitriptyline, which might include gastrointestinal symptoms, affective symptoms, general somatic symptoms, and sleep disturbance (Haddad and Anderson 2007). However, the longer the treatment lasts and the higher the dosage of antidepressant, the more likely discontinuation symptoms occur (Kramer et al. 1961; Perahia et al. 2005). Both of these circumstances do not apply to our study, meaning that the explanation that differences between the amitriptyline and placebo group at the evocation night were only due to discontinuation effects is not likely. Despite these limitations, our study encourages future research to examine Pavlovian conditioning in conjunction with side effects. To learn more about these mechanisms, future studies should vary the number of learning trials and of the intervals between acquisition and evocation. It would be critical to determine if and when conditioned effects extinguish when the interval between acquisition and evocation is long enough. In addition, we only examined the effect associated with one drug; thus, the learning of side effects should also be addressed in conjunction with other drugs. To draw conclusions for pharmacotherapy in clinical settings, patients rather than healthy individuals need to be examined. Understanding the mechanisms that lead to the learning of side effects may help to prevent the side effects triggered by prior experience. Several proposals about how to reduce side effects have been made (Colloca and Miller 2011; Bingel 2014). Such interventions focus mainly on the expectations of side effects induced by verbal suggestion or other information and how to modify them to minimize side effects (von Blanckenburg et al. 2013). As our findings suggest that prior negative experience with a drug can also lead to side effects and hence non-adherence, it is important to develop additional strategies to prevent these learned side effects.

TITLE: Exogenous dopamine reduces ABSTRACT.ABSTRACT.BACKGROUND: While it has recently been shown that dopamine release stimulates conscious self‐monitoring through the generation of gamma oscillations in medial prefrontal/anterior cingulate cortex, and that the GABAergic system is effective in producing such oscillations, interaction of the two transmitter systems has not been demonstrated in humans. We here hypothesize that dopamine challenge stimulates the GABA system directly in the medial prefrontal/anterior cingulate region in the human brain. ABSTRACT.ABSTRACT.METHODS: Positron emission tomography (PET) with the GABA receptor α1/α5 subtype ligand [11C] Ro15‐4513 was used to detect changes in GABA receptor availability after clinical oral doses of levodopa in a double blind controlled study. ABSTRACT.ABSTRACT.RESULTS: We here provide the first direct evidence for such coupling in the cerebral cortex, in particular in the medial prefrontal anterior cingulate region, by showing that exogenous dopamine decreases [11C] Ro15‐4513 binding widely in the human brain compatible with a fall in α1 subtype availability in GABA complexes due to increased GABA activity. BODY.INTRODUCTION: Cognition depends on paralimbic medial prefrontal/anterior cingulate and medial parietal hubs (Lou et al. 2004; Luber et al. 2012) and their rich network of highly oxygen‐demanding, fast‐spiking GABAergic interneurons (Tomasi et al. 2013). The network is regulated by dopamine, which in humans stimulates medial prefrontal/anterior cingulate cortex and insula, thereby generating gamma oscillations (Joensson et al. 2015) essential for cognitive function (Voss et al. 2014). The high oxygen requirement of these hubs (Tomasi et al. 2013) makes the system vulnerable in penuria, and dysfunction is associated with disorders like ADHD (Castellanos et al. 2008), autistic spectrum disorder (Assaf et al. 2010), schizophrenia (Gallinat et al. 2004), addiction (Romer et al. 2013), and dementia (Liu et al. 2014), and can follow traumatic brain injury (Ham et al. 2014). It is, therefore, important to determine how the information gathered in animal models on the molecular arrangement of dopamine/GABA interaction (Sesack et al. 2003) translates into the human brain. This is particularly the case for the medial prefrontal/anterior cingulate region which facilitates attention and self‐awareness. With that aim we here examined the effect of oral l‐dopa, a dopamine prodrug, on the activation of the GABA system in the human living cerebral cortex. The idea that dopamine release regulates cognition, including conscious experience, via the GABAergic system is based on the finding that dopamine increases confidence and accuracy of seeing rapidly presented words (Lou et al. 2011), and on the fact that GABAergic interneurons are instrumental in mediating cognition in rodents (Sohal et al. 2009). The hypothesis is further strengthened by the recent demonstration that dopamine release in the medial prefrontal/anterior cingulate cortex activates self‐monitoring and metacognition (Joensson et al. 2015). Theoretical aspects of interaction between dopamine and GABA function have been studied in detail in a recent neurocomputational model by Lew and Tseng (2014). Direct experimental approaches have been scarce, but recently it has been demonstrated that extracellular striatal dopamine elicits opposing effects on the GABA system in rodents: dopamine release activates synaptically located GABAA receptor subtypes, including alpha 5, but inhibits tonic GABAA currents via extrasynaptically located tonic level GABAA receptors (Hoerbelt et al. 2015). These effects of dopamine mimic similar opposing effects of extracellular GABA (Ransom et al. 2013), and are in accordance with the concept that tonic GABA currents are a homeostatic regulator responding to localized synaptic stimulation of GABA receptors by decreasing excitability in the network (Stokes et al. 2014). Here, we provide the first direct in vivo evidence for such interaction in humans by showing that an exogenous dopamine challenge in physiological relevant dosage decreases GABA receptor availability, reflecting increased GABA activity. BODY.PARTICIPANTS AND PROCEDURES: The study was approved by the local ethical board (Region Midtjylland). After information given orally and in writing, and consent, nine healthy males were studied. This provides 80% power to detect a 20% reduction of anterior cingulate Ro 15‐4513 binding at P = 0.05. The participants were without current medication. None had a history of mental or neurological disorders, or a history of addiction, with the exception that two had been smokers 10 and 15 years ago, respectively. Median age was 29 years, range 24–47. The PET ligand [11C]Ro15‐4513 is sensitive to interstitial GABA levels evidenced as a fall in availability of α1 subtype sites on the GABA complex for its binding (Semyano et al. 2004; Stokes et al. 2014). While [11C]Ro15‐4513 binds to both the α1 and α5 receptor subtypes, the α1 subtype is present at a greater density and increases in brain GABA have been shown to reduce its availability, while α5 binding site availability is either unaffected or mildly elevated. In the present study, it was not possible to separate signals from α1 and α5 subunits of the GABA complex as we did not have an arterial input curve so we have assumed that any decreased Ro 15‐4513 binding seen represented competitive occupancy of α1 sites by endogenous GABA (Lingford‐Hughes et al. 2002). Each participant was studied twice in counterbalanced order with an interval of at least several days. We used PET following an oral l‐dopa/dopamine challenge or placebo in identical capsules given orally containing either sinemet (MSD, 100 mg l‐dopa plus 25 mg carbidopa) or placebo (starch). The medication was unknown to the participants and experimenters responsible for PET injection and calculation of binding potentials. The administration of l‐dopa or placebo was done between 30 and 45 min prior to intravenous injection of [11C]Ro15‐4513. This interval is based on the interval for obtaining peak plasma concentrations and clinical effects on parkinsonism after oral sinemet medication (Joensson et al. 2015). BODY.RESULTS AND DISCUSSION: The distribution of [11C]Ro15‐4513 binding with placebo is shown in Figure 1. After placebo administration, the ligand is bound primarily in medial prefrontal/anterior cingulate cortex and left and right insula. The cerebellum provides a reference for nonspecific tissue binding of the tracer. After l‐dopa, [11C]Ro15‐4513 binding is reduced, which can be seen more clearly by subtraction. There is a reduction of between 5% and 20% throughout most of gray matter regions (BP 100x [placebo minus dopamine]/placebo). Small foci of BP increases were, however, present in the hippocampi, possibly reflecting the high concentration of D4 dopamine receptors there which inhibit interneuron GABA release when activated (Romo‐Parra et al. 2005). Figure 1Distribution of [11C]Ro15‐4513. After placebo, the ligand was bound primarily in medial prefrontal/anterior cingulate cortex and left and right insula. After dopamine challenge ligand binding is reduced. This is seen more clearly by subtraction, with a general reduction of between 5% and 20% throughout gray matter (BP 100x [placebo minus dopamine]/placebo). In the hippocampal regions there were small foci of increased BP. Quantification using parametric modeling is shown in Figure 2 for medial prefrontal/anterior cingulate region, left and right insula, cerebellum, and whole gray matter. For a composite gray matter region, a one‐sample t‐test showed that l‐dopa was associated with an 11% mean decrease in [11C]Ro15‐4513 BP (t = −2.29, 9 degrees of freedom, P = 0.048). There was a highly significant effect of regions (linear mixed model, P = 0.000049). Because dopamine has previously been shown to stimulate electromagnetic activity and self‐awareness mainly in the medial prefrontal/anterior cingulate and insula regions (Joensson et al. 2015), we a priori calculated BP changes in these regions separately and compared these with cerebellum and whole gray matter, using two‐tailed t tests with Bonferroni corrections and a cut off at P = 0.01. The BP of the GABA ligand decreased significantly with l‐dopa in the medial prefrontal/anterior cingulate and right insula regions (by 18%, t = −3.63 P = 0.0054, and 18%, t = 3.85 P = 0039, respectively), with no significant effect in the other regions. Figure 2Quantification in medial prefrontal/anterior cingulate region, left and right insula, cerebellum, and whole gray matter. Using parametric modeling, a decrease in [11C]Ro15‐4513 BP is apparent overall after l‐dopa, except in cerebellum. For statistics, see text. Thus, the main findings were (1) preferential binding of [11C]Ro15‐4513 to α1/α5 subunits on GABA complexes in the medial prefrontal/anterior cingulate region and (2) a widespread and proportional dopamine‐induced decrease in binding potentials for the GABA ligand [11C]Ro15‐4513 (Lingford‐Hughes et al. 2002). The present results account for the effect of dopamine in its regulation of self‐awareness via direct action on the GABA system in the medial prefrontal/anterior cingulate cortex. Similarly, the direct effect of dopamine on GABA in right insula, rather than the left, may explain the preferential role of right hemisphere in self‐recognition (Keenan et al. 2001). The close cortical interaction of the two transmitter systems broadens the scope for pharmacological intervention in the many human disorders where the medial prefrontal region is dysfunctional. It may open new perspectives for treatment. BODY.METHODS.MAGNETIC RESONANCE IMAGING: All participants were scanned with a 3T MR system pre‐registration with PET (Siemens Trio, Erlangen, Germany). A T1 MPRAGE scan (TR/TE 2420/3.7 msec, 1 mm isotropic resolution, scan time 51⁄2 min) was performed. BODY.METHODS.SYNTHESIS AND PURITY OF [: [11C]15‐4513 was synthesized by the adoption of the procedure of Halldin et al. (1992). Briefly, 11CO2, either produced with the PET centers GE PETtrace or IBA18/18 cyclotron, was converted to [11C]methyl iodide applying a GE Healthcare MeI Microlab. [11C]methyl iodide was trapped at room temperature in a solution of 0.3–0.5 mg Ro44‐3902 (ABX advanced biochemical compounds, Radeberg, Germany) in 0.3 mL DMF, containing 0.5–1 mg NaH (60% dispersion in mineral oil; Sigma‐Aldrich A/S, Brøndby, Denmark). After heating for 1 min at 50°C [11C]15‐4513 was isolated by preparative HPLC applying a 250 × 10 mm Phenomenex Luna C18(2) column and a mixture of 70 mmol/L NaH2PO4 buffer and acetonitrile (70:30). The fraction containing [11C]15‐4513 was collected, diluted with 50 mL of sterile water. Afterward [11C]15‐4513 was trapped on Water Sep‐Pak A/S, Hedehusende, Denmark® C‐18 Plus light cartridge. Remaining acetonitrile was removed by flushing the Sep‐Pak cartridge with 20 mL of sterile water. [11C]15‐4513 was desorbed with 1 mL of sterile ethanol followed by 9 mL of saline. The final solution was sterile filtered via 0.22‐μm sterile filter into a sterile product vial. This procedure yielded 1–6 GBq of [11C]15‐4513 within 35 min and radiochemical purities >95%. Amounts of nonradioactive [12C]15‐4513 were in the range 0.3–3.5 μg/mL (1–11 nmol/mL). Interestingly, we found increased radiolysis on the C‐18 Sep‐Pak affecting the radiochemical purity at very high radioactivities. BODY.METHODS.POSITRON EMISSION TOMOGRAPHY: Participants were placed in the scanner with the orbitomeatal line parallel to the transaxial plane of the tomograph. Head position was monitored via laser crosshairs and video camera. In order to correct for attenuation of emitted radiation by skull and tissues, a transmission scan was acquired using a single rotating photon point source of 150 MBq of 137Cs. Participants were pretreated with placebo or 100 mg l‐dopa + 25 mg carbidopa given orally to block peripheral metabolism of l‐dopa, thereby increasing tracer delivery to the brain. This was done 30–45 min prior to injection of 400 (range 375–425) MBq [11C]Ro15‐4513 through a cubital vein in 10 mL of normal saline over 30 sec. Three‐dimensional PET was recorded over 60 min, using an ECAT EXACT HR++ (CTI/Siemens 966; Siemens PET/CT Biograph, Erlangen, Germany) camera, which covers an axial field of view of 23.4 cm and provides 95 transaxial planes. The tomograph has a spatial resolution of 4.8 + 0.2 mm FWHM (transaxial, 1 cm off axis) and 5.6 mm + 0.5 mm (axial, on axis) after image reconstruction. PET and MRI were coregistered to a common MR T1 atlas from Montreal Neurological Institute (MNI) (Collins et al. 1998) using PMOD (PMOD Technologies Ltd., Zurich, Switzerland). The kinetic parameters of [11C]Ro15‐4513 were determined by the SRTM2 method using cerebellum as reference (Wu and Carson 2002). No FDR or cluster corrections were used as the regions were predefined. BODY.CONFLICT OF INTEREST: The authors declare no competing financial interest.

TITLE: Messaging to Increase Public Support for Naloxone Distribution Policies in the United States: Results from a Randomized Survey Experiment ABSTRACT.BACKGROUND: Barriers to public support for naloxone distribution include lack of knowledge, concerns about potential unintended consequences, and lack of sympathy for people at risk of overdose. ABSTRACT.METHODS: A randomized survey experiment was conducted with a nationally-representative web-based survey research panel (GfK KnowledgePanel). Participants were randomly assigned to read different messages alone or in combination: 1) factual information about naloxone; 2) pre-emptive refutation of potential concerns about naloxone distribution; and 3) a sympathetic narrative about a mother whose daughter died of an opioid overdose. Participants were then asked if they support or oppose policies related to naloxone distribution. For each policy item, logistic regression models were used to test the effect of each message exposure compared with the no-exposure control group. ABSTRACT.RESULTS: The final sample consisted of 1,598 participants (completion rate: 72.6%). Factual information and the sympathetic narrative alone each led to higher support for training first responders to use naloxone, providing naloxone to friends and family members of people using opioids, and passing laws to protect people who administer naloxone. Participants receiving the combination of the sympathetic narrative and factual information, compared to factual information alone, were more likely to support all policies: providing naloxone to friends and family members (OR: 2.0 [95% CI: 1.4 to 2.9]), training first responders to use naloxone (OR: 2.0 [95% CI: 1.2 to 3.4]), passing laws to protect people if they administer naloxone (OR: 1.5 [95% CI: 1.04 to 2.2]), and passing laws to protect people if they call for medical help for an overdose (OR: 1.7 [95% CI: 1.2 to 2.5]). ABSTRACT.CONCLUSIONS: All messages increased public support, but combining factual information and the sympathetic narrative was most effective. Public support for naloxone distribution can be improved through education and sympathetic portrayals of the population who stands to benefit from these policies. BODY.INTRODUCTION: Between 1999 and 2013, the number of people dying from a drug overdose involving opioid analgesics in the United States more than quadrupled, from 4,030 to 16,235 [1]. More recently, an increase in heroin overdose deaths has been observed in several states, with some areas noting a marked transition from overdose deaths involving opioid analgesics to heroin overdoses [2–4]. Multiple strategies to address opioid overdose mortality have been proposed, including naloxone distribution programs [5]. Naloxone, an opioid antagonist, has long been used in medical settings and is highly effective in reversing the effects of an opioid overdose [6]. More recently, numerous programs across the United States have distributed naloxone, paired with education on how to identify, prevent, and treat overdoses, to the lay public (e.g., friends and family members of people who use opioids). Evaluations of these programs have consistently found that lay persons can accurately identify and respond to an opioid overdose with intranasal or intramuscular naloxone, preventing a fatal overdose [7–16]. According to a report by the Centers for Disease Control and Prevention, between 1996 and 2010 over 50,000 people in the United States were trained to use naloxone, resulting in over 10,000 reported overdose reversals [17]. On a community level, recent research has found strong associations between naloxone distribution programs and lower rates of opioid overdose fatalities [18]. In addition to naloxone distribution programs for friends and family members of people who use opioids, programs training first responders like police, firefighters, and emergency medical technicians who traditionally have not been trained to use naloxone, have also demonstrated positive results [19,20]. Despite evidence of safety and efficacy, naloxone distribution programs, like other harm reduction interventions, face logistical and ideological challenges [21,22]. First, individuals who witness an overdose may not call for medical attention or administer naloxone because of legal concerns [23–25]. Medical providers that prescribe naloxone may have similar concerns [25–27]. To address this issue, laws protecting individuals who call for medical attention for an overdose as well as laws protecting individuals who administer, and providers who prescribe, naloxone have been enacted in several states [26,28,29]. Second, some critics argue that giving naloxone to people who use opioids could potentially lead to increased use because people will rely on naloxone to rescue them from a life-threatening overdose [21]; however, this concern is not supported by previous research. In fact, some research suggests that opioid use may be reduced after participating in a naloxone distribution program as these programs provide education on how to prevent an overdose [8,10,12]. Third, some critics argue that preventing overdose mortality is futile because people who use opioids will continue using and overdose again. While opioid use disorder is a chronic medical illness with periods of remission and relapse, treatment does improve outcomes [30–33], and individuals who experience a fatal overdose have lost the opportunity to engage in it. Although data on public opinion about naloxone distribution programs are lacking, there are many potential reasons why naloxone distribution may have a low rate of public support. Stigma and negative public opinion around drug use and people who use drugs are barriers to support of a wide range of public policies such as insurance parity, housing support, and job support [34]. More specific to naloxone distribution, lack of familiarity with naloxone, concerns about the unintended consequences of naloxone, and lack of compassion or sympathy for people who use drugs may lead to low public support. Previous research shows that public opinion is a substantial contributor to the enactment of public policy [35]. For example, stigma and negative public opinion toward people who use drugs are connected to the criminalization of drug use and enactment of punitive criminal justice-focused policies [36]. Improving public support for naloxone distribution may contribute to wider implementation. If there is a general gap in knowledge about the effects of naloxone distribution, factual information about the safety and efficacy of naloxone and pre-emptive refutation of potential objections to naloxone distribution might improve public support [37]. In addition, as many Americans view drug use as a moral failing [36], overdose may be viewed as the predictable consequence of poor choices as opposed to the result of a medical condition warranting public health intervention; presenting narratives which evoke emotional responses such as sadness or sympathy may improve public support for naloxone distribution in this case [38,39]. To examine the effects of different types of messages to improve public support for, and foster positive beliefs about, naloxone distribution in addition to other policies aimed at reducing opioid overdose mortality, we conducted a randomized survey experiment. We hypothesized that provision of factual information alone would increase support for naloxone distribution, but it would also increase negative beliefs about the unintended consequences of naloxone distribution. We also hypothesized that pre-emptive refutation of these concerns would prevent increases in negative beliefs. Finally, we hypothesized that a sympathetic narrative about naloxone's benefits would also bolster support, and the pairing of factual information with a sympathetic narrative would be most effective at increasing support for naloxone distribution policies. BODY.METHODS: A randomized survey experiment was fielded September 18th through October 13th, 2014, using the nationally-representative web-based GfK survey research panel (KnowledgePanel). The survey panel consists of about 55,000 adult members ages 18 and older who were recruited through address-based sampling using a frame of residential addresses that covers approximately 97% of US households [40]. Recruited households without Internet access were provided with laptop computers. Newly recruited panel members complete a demographic profile including information such as gender, age, race/ethnicity, income, education. Survey participation was rewarded with a variety of small incentives (small cash awards, gift prizes, sweepstakes opportunities), and on average, panel members participate in about two surveys per month. In the current study, invitations to participate did not include information about survey content. To account for nonresponse and error from panel recruitment methods or panel attrition, survey weights were calculated and provided by GfK along with survey responses. The GfK panel has been used extensively for survey research in diverse academic disciplines [41–45]. BODY.METHODS.MESSAGE CONTENT: We tested the effects of three different types of persuasive messages on public support for naloxone distribution programs, alone or in combination: (1) factual information (text about opioid analgesic overdoses and evidence on the safety and efficacy of naloxone in preventing overdose death); (2) pre-emptive refutation (text providing counterarguments to two common concerns around naloxone distribution, that having access to naloxone will lead to more overdoses because people will believe they can be "rescued" and that people who overdose and are saved with naloxone will just continue using and overdose again); and a (3) sympathetic narrative (text about a mother who struggles with her daughter's addiction to opioid analgesics and subsequent fatal overdose). The factual information message was 13 sentences in length, the pre-emptive refutation message was 8 sentences in length, and the sympathetic narrative was 20 sentences in length. The sympathetic narrative was longer than the factual information and pre-emptive refutation messages because effective stories require contextual information to humanize characters and offer a compelling storyline. Prior to reading the randomized message(s), all participants read a brief definition of opioid analgesics—termed "prescription pain medication" throughout the survey to be more accessible to participants—which included a link to view a medication list (S1 Appendix). The factual information read as follows: In 2011, nearly 17,000 people died from prescription pain medication overdoses in the United States—equal to 46 deaths per day. Over the last decade, the number of prescription pain medication overdose deaths has increased by more than 300 percent. Government officials, medical experts, and community leaders have declared prescription pain medication overdoses a national crisis. Naloxone is a medicine that is very effective at saving lives by reversing life threatening overdoses of prescription pain medication. Naloxone can be given by injection or nasal spray. Medical experts believe naloxone is so safe that anyone can be trained to administer it, even friends and family members of people at risk of overdose and first responders like police officers and firefighters. If naloxone is mistakenly given to someone who is not having a prescription pain medication overdose, there are no bad side effects. Most overdoses occur among people who are at home with friends or family. If these friends or family members had naloxone, they could administer it to potentially save the life of a person overdosing. When someone overdosing is using prescription pain medication illegally, friends and family are often afraid to call the police because they don't want to be arrested and put in jail for being around someone using drugs illegally. Even if they do call, most police officers and firefighters do not have naloxone and have to wait for an ambulance to arrive. A person overdosing may die before getting the naloxone treatment he or she needs. Providing training and naloxone medication to friends and family members of people at risk of overdose and first responders like police officers and firefighters could save thousands of lives every year. The pre-emptive refutation message read as follows: Some people don't believe that the lives of people overdosing on prescription pain medication are worth saving. They say that using naloxone to save a person overdosing is pointless because the person will just continue using prescription pain medication and eventually overdose again. Some people also say that giving naloxone to people who are addicted to prescription pain medication will just cause them to use more, because they will think of naloxone as a "safety net" to save them from an overdose. But in fact, many people who overdose and are saved because of naloxone will see it as a wake-up call and enter treatment for their addiction. Letting someone die from an overdose that could be prevented is cruel and misguided, especially because naloxone is such a safe medication. Friends and family members of people addicted to prescription pain medication often feel helpless watching their loved one struggle, but providing naloxone to these friends and family members is giving them the power to save a life. And providing naloxone to first responders like police officers and firefighters allows them to be better prepared to help when they arrive at the scene of an overdose. People saved from a prescription pain medication overdose can recover and go on to live long, productive, and healthy lives. The sympathetic narrative read as follows: Mother's Day has become a very difficult time for Mary since she lost her daughter, Erika, to an overdose of prescription pain medication two years ago. It all started after Erika was hit by a car while driving home from college. Left with back, hip, and knee injuries and severe pain, Erika turned to doctors for help. She started physical therapy and her doctor prescribed Percocet, Vicodin, and OxyContin—strong prescription pain medications—to help ease her pain. For the first few months, things were going well. Erika was recovering her ability to get around and was catching up on her school work. But then, Mary saw something change. Erika started taking more prescription pain medication. When her prescription ran out and the doctor would not give her another, she started getting old prescriptions from friends. Mary suspected that Erika had developed an addiction to prescription pain medication and tried to convince her to get help. At first Erika said she wasn't addicted and didn't need help, but after a few more months she admitted to her mother that she had a problem. Even though Erika was willing to get help, Mary couldn't find an addiction treatment program that was seeing new patients anywhere in her community. She finally found a clinic nearly two hours away that could treat Erika, but the first available appointment was several weeks away. Mary scheduled the appointment, but a few days later she came home from work and found Erika on the bathroom floor, barely breathing. Mary called 911, but by the time Erika got to the hospital it was too late and she was pronounced dead from an overdose of pain medication. Thinking back, Mary wishes she had known about naloxone, a medication she could have been trained to use in an emergency that helps people who are overdosing. Naloxone could have saved her daughter's life. Mary recently got trained to use naloxone and started a support and education group for parents who have children that are addicted to prescription pain medication. She has also been pushing for the local police and fire departments in her town to train first responders to carry naloxone medication in case they arrive at the scene of an overdose before paramedics. On top of her full-time job, Mary has been working tirelessly to prevent overdose deaths in her community because she believes no parent should have to go through losing a child the way she did. We randomized survey participants to a no-exposure control group or to read one of five message combinations: (1) factual information only, (2) factual information plus pre-emptive refutation, (3) sympathetic narrative only, (4) sympathetic narrative plus factual information, or (5) all three messages in combination. BODY.METHODS.OUTCOME MEASURES: We examined effects of randomized condition on two main categories of outcomes created by the study team: support for naloxone distribution and other overdose mortality prevention policies and beliefs about naloxone. For naloxone distribution and overdose mortality prevention policies, we asked participants whether they support or oppose:1) training first responders like police officers and firefighters to use naloxone in cases when they arrive at the scene before paramedics; 2) providing naloxone to friends and family members of people using opioid analgesics; 3) passing laws to protect people who call for medical help when they themselves or someone else is experiencing an overdose; 4) passing laws to provide legal protection for people giving naloxone to a friend or family member; and 5) increasing government spending to improve screening and treatment of opioid addiction. For beliefs about naloxone, we asked if participants agreed or disagreed that: 1) providing naloxone to first responders, like police officers and firefighters, would save lives; 2) providing naloxone to friends and family members of people who use opioid analgesics would save lives; 3) distributing naloxone will encourage people to use even more opioid analgesics because they will assume they can be saved from a life-threatening overdose; 4) preventing overdoses is ineffective because people with opioid addiction will continue to use and eventually overdose again; and 5) naloxone is a medication that should only be given by medical professionals. We randomized the order of policy and belief question blocks for each survey participant, as well as the order of questions within the blocks, to cancel out any effects of question order on responses. We asked each question using seven-point scales anchored at 1 (strongly oppose), 4 (neither oppose nor favor), and 7 (strongly favor) for policy questions and 1 (strongly disagree), 4 (neither agree nor disagree), and 7 (strongly agree) for belief questions. We did not include a "don't know" option, but participants could choose not to answer a given question. For policy questions, we dichotomized responses into support (5–7) or do not support (1–4) and for belief questions, we dichotomized responses into agree (5–7) or do not agree (1–4). Responses were dichotomized in this way as 50% is a meaningful cut-off for policy support (majority support) and thus modeling the outcomes dichotomously allowed for estimation of the predicted probabilities of majority support. We excluded survey responses if participants completed the survey in fewer than 1.5 minutes (1 minute for the no message control group) or greater than 240 minutes. Of the 2,321 panel members invited to respond, 1,685 completed the survey for a completion rate of 72.6%. Among participants, 5.2% (87/1,685) were excluded for not completing the survey in the specified time window. BODY.METHODS.STATISTICAL ANALYSIS: To assess the representativeness of survey participants, we examined socio-demographic characteristics (age, gender, race/ethnicity, education level, employment status, geographic region of residence) using the un-weighted and weighted responses in comparison to national estimates from the March 2013 Current Population Survey. Characteristics of the study sample in terms of gender, age, race, education level, household income, employment status and region were close to the national comparison (Table 1). There was an overrepresentation in the unweighted study sample of white people (+ 7.5%), unemployed people (+ 2.6%), and those age 55 to 64 years (+ 5.9%). Next, to ensure randomization was successful at producing balanced groups, we compared the socio-demographic characteristics of participants in each survey arm using chi-square tests. Randomization to the experimental conditions resulted in six well balanced groups without any significant differences in the above mentioned characteristics (Table 2). Finally, we calculated the percentage of participants supporting each policy item or agreeing with each belief, along with 95% confidence intervals (95% CI). The item response rate was 99.5% and for a given question, participants with missing responses were excluded from that question but included in all other questions for which data were available. 10.1371/journal.pone.0130050.t001 Table 1 Un-weighted and weighted characteristics of survey participants compared with national rates (N = 1,598). Un-weighted Weighted National Comparison Women (%) 50.1 52.0 51.9 Age (%)     Ages 18–24 10.1 12.3 12.7     Ages 25–34 16.2 17.6 17.5     Ages 35–44 15.8 16.3 16.8     Ages 45–54 16.5 16.1 18.4     Ages 55–64 22.2 20.4 16.3     Age 65 + 19.2 17.3 18.3 Race (%)     White only 73.5 66.1 66.0     Black only 9.4 11.5 11.6     Other 17.2 22.5 22.5 Hispanic ethnicity (%) 10.0 15.1 15.0 Education (%)     < High school degree 9.9 12.4 12.6     High school degree 31.5 29.8 29.6     Some college 27.6 28.9 28.9     Bachelor's degree or higher 31.0 28.9 28.9 Household income (%)     Under $10,000 5.3 5.8 5.2     $10,000–24,999 12.9 11.9 13.3     $25,000–49,999 21.8 22.7 22.7     $50,000–74,999 19.2 18.1 18.4     $75,000 or higher 40.9 41.6 40.5 Employment status (%)     Employed 57.2 57.9 59.9     Unemployed 7.5 8.2 4.9     Retired 19.1 16.9 17.2     Other (e.g., disabled, homemaker, other) 16.3 17.0 18.1 Region (%)     Northeast 18.5 17.8 18.2     Midwest 23.2 21.7 21.4     South 36.1 37.1 37.1     West 22.2 23.5 23.4 Note: GfK sample weights used to calculate descriptive statistics. For socio-demographic characteristics, comparison data extracted from the March 2013 Current Population Survey. 10.1371/journal.pone.0130050.t002 Table 2 Comparison of the characteristics of survey participants randomized to each message exposure (N = 1,598). No-exposure control (n = 267) Factual information (n = 260) Factual information plus refutation (n = 266) Sympathetic narrative (n = 264) Sympathetic narrative plus factual information (n = 276) Sympathetic narrative plus factual information plus refutation (n = 265) P Women (%) 52.6 51.3 51.8 51.1 52.2 52.9 0.99 Age (%) 0.98     Ages 18–24 13.1 13.5 12.2 10.5 11.5 13.3     Ages 25–34 15.9 15.3 17.1 21.4 19.0 16.9     Ages 35–44 17.9 18.1 16.4 14.5 16.5 14.6     Ages 45–54 15.8 15.3 15.7 14.5 18.1 16.9     Ages 55–64 19.1 21.1 21.3 24.1 18.4 18.7     Age 65 + 18.2 16.8 17.3 15.0 16.6 19.7 Race (%) 1.0     White only 64.8 65.5 66.2 66.9 66.9 66.1     Black only 11.8 11.3 11.2 11.7 11.5 11.2     Other 23.5 23.2 22.6 21.4 21.7 22.7 Hispanic ethnicity (%) 15.5 15.4 14.8 14.8 15.2 14.9 0.99 Education (%) 1.0     < High school degree 12.7 12.2 12.0 12.9 12.2 12.4     High school degree 29.5 29.6 30.4 29.4 29.9 30.1     Some college 29.3 29.0 28.1 27.5 29.4 30.0     Bachelor's degree or higher 28.6 29.2 29.5 30.3 28.5 27.5 Household income (%) 0.99     Under $10,000 6.1 4.8 3.9 6.9 6.1 7.2     $10,000–24,999 11.8 13.3 14.0 10.4 11.1 10.8     $25,000–49,999 22.2 22.0 22.6 22.9 22.9 23.3     $50,000–74,999 18.1 18.6 18.0 18.6 18.7 16.6     $75,000 or higher 41.8 41.3 41.6 41.2 41.3 42.1 Employment status (%) 0.48     Employed 56.3 59.6 62.3 55.5 59.2 54.3     Unemployed 6.2 7.2 9.5 9.4 6.3 10.9     Retired 16.8 17.0 16.4 15.2 17.3 18.8     Other (e.g., disabled, homemaker, other) 20.8 16.2 11.9 19.9 17.2 16.0 Region (%) 1.0     Northeast 17.6 18.1 17.8 18.9 16.8 17.3     Midwest 21.8 21.3 21.6 22.2 22.2 21.2     South 37.9 37.1 36.7 36.3 37.5 36.8     West 22.7 23.6 23.8 22.7 23.5 24.7 To test the effect of each message exposure compared with the no-exposure control group, we then created logistic regression models for each outcome question. In each model, the dependent variable was support for a specific policy or agreement with a specific belief and the main independent variable was the randomized message exposure. Using the dichotomized measures of policy support and agreement with beliefs, we constructed odds ratios (OR) with 95% CIs to compare the effect of each message exposure to the no-exposure control group. Next, to examine the effects of adding the sympathetic narrative to factual information, we used similar logistic regression models to compare the effect of exposure to the sympathetic narrative plus factual information, using participants exposed to factual information alone as the referent group. As survey participants were randomly assigned to message exposures, we did not include covariates in regression models [46]. We conducted all statistical analyses using Stata 13.1(StataCorp LP, College Station, TX, USA). BODY.METHODS.SENSITIVITY ANALYSIS: As a sensitivity analysis, we created separate policy support and belief scales from the policy questions and the belief questions, respectively. We used Crohnbach's alpha to assess consistency of the items and used factor analysis to identify candidates for deletion. After reverse coding the negatively worded questions, we averaged responses to policy questions and belief questions to create the scales. Finally, we created linear regression models to determine the effect of different messages on the scales. For each model, the policy support or belief scale was the dependent variable and the message exposure was the main independent variable. BODY.METHODS.ETHICS STATEMENT: This study was determined to be exempt by the Johns Hopkins Bloomberg School of Public Health Institutional Review Board. BODY.RESULTS.SUPPORT FOR NALOXONE DISTRIBUTION AND OTHER POLICIES IN THE NO-EXPOSURE CONTROL GROUP: The first column of Table 3 indicates support for overdose mortality prevention policies among the no-exposure control group. Public support was strongest for training first responders to use naloxone (63.2%) and passing laws to protect people if they call for medical help for an overdose (52.4%). Fewer participants in the no-exposure control group supported policies to protect people from legal action if they administer naloxone to friends or family members (41.6%) or increasing government spending to improve addiction screening and treatment (38.5%). Public support was lowest for the policy to provide naloxone to friends and family members of people who use opioid analgesics (24.4%). 10.1371/journal.pone.0130050.t003 Table 3 Effects of messages on support for naloxone distribution and other overdose mortality prevention policies compared to the no-exposure control group. Message Exposure Policy a No-exposure control (n = 267) Factual information (n = 260) Factual information plus refutation (n = 266) Sympathetic narrative (n = 264) Sympathetic narrative plus factual information (n = 276) Sympathetic narrative plus factual information plus refutation (n = 265) Train first responders to use naloxone (n = 1592) 63.2% (56.9 to 69.1) 77.4% (71.2 to 82.6) ** 79.0% (73.3 to 83.7) *** 80.5% (74.7 to 85.2) *** 87.4% (82.5 to 91.1) *** 85.7% (80.0 to 89.9) *** Provide naloxone to friends and family members (n = 1592) 24.4% (19.4 to 30.4) 45.5% (39.2 to 55.4) *** 49.0% (42.7 to 55.4) *** 40.1% (34.2 to 46.4) *** 62.6% (56.4 to 68.4) *** 64.3% (57.6 to 70.5) *** Pass laws to protect people if they call for medical help for an overdose (n = 1592) 52.4% (45.9 to 58.7) 53.3% (46.8 to 59.8) 59.0% (52.6 to 62.2) 58.0% (51.7 to 64.1) 66.5% (60.2 to 72.2) ** 69.2% (62.7 to 75.0) *** Pass laws to protect people if they give naloxone (n = 1592) 41.6% (35.4 to 48.0) 58.0% (51.4 to 64.3) *** 58.6% (52.1 to 64.8) *** 55.6% (49.3 to 61.7) ** 67.7% (61.5 to 73.3) *** 69.1% (62.4 to 75.0%) *** Increase government spending to improve addiction screening and treatment (n = 1587) 38.5% (32.3 to 45.0) 33.9% (28.1 to 40.2) 34.3% (28.5 to 40.6) 47.9% (41.7 to 54.2) * 50.6% (44.3 to 56.9) ** 43.2% (36.7 to 49.8) *P ≤ 0.05 compared to the no-exposure control group using logistic regression ** P ≤ 0.01 compared to the no-exposure control group using logistic regression *** P ≤ 0.001 compared to the no-exposure control group using logistic regression a Percent support for policies calculated as percentage of sample responding 5, 6, or 7 on the seven point scale for each measure (i.e., somewhat favor, favor, or strongly favor) BODY.RESULTS.EFFECTS OF MESSAGES ON SUPPORT FOR NALOXONE DISTRIBUTION AND OTHER POLICIES: Several message exposures led to significantly higher support for policies compared with the no-exposure control group (Table 3, Fig 1 and S1 Fig). Factual information alone led to higher support for training first responders to use naloxone (77.4% versus 63.2% in the no-exposure control group), providing naloxone to friends and family members of people using opioids (45.5% versus 24.4% in the no-exposure control group), and passing laws to protect people who administer naloxone (58.0% versus 41.6% in the no-exposure control group). The sympathetic narrative alone also led to higher support for training first responders to use naloxone (80.5% versus 63.2% in the no-exposure control group), providing naloxone to friends and family members of people using opioids (40.1% versus 24.4% in the no-exposure control group), passing laws to protect people who administer naloxone (55.6% versus 41.6% in the no-exposure control group), and increasing government spending to improve addiction screening and treatment (47.9% versus 38.5% in the no-exposure control group). Results from the sensitivity analysis using a policy support scale were similar (S2 Appendix). 10.1371/journal.pone.0130050.g001Fig 1Effects of factual information, a sympathetic narrative, or both on support for naloxone distribution and other opioid overdose mortality prevention policies. Directly comparing the effects of adding the sympathetic narrative to factual information, using participants exposed to factual information alone as the referent group, we found that the combination led to higher support for all policies: providing naloxone to friends and family members (OR: 2.0 [95% CI: 1.4 to 2.9]), training first responders to use naloxone (OR: 2.0 [95% CI: 1.2 to 3.4]), passing laws to protect people if they administer naloxone (OR: 1.5 [95% CI: 1.04 to 2.2]), passing laws to protect people if they call for medical help for an overdose (OR: 1.7 [95% CI: 1.2 to 2.5]), and increasing government spending to improve addiction screening and treatment (OR: 2.0 [95% CI: 1.4 to 2.9]). BODY.RESULTS.BELIEFS ABOUT NALOXONE IN THE NO-EXPOSURE CONTROL GROUP: The first column of Table 4 shows agreement with beliefs about naloxone among participants in the no-exposure control group. A majority believed that providing naloxone to first responders would save lives (60%); however, only 35.9% believed that providing naloxone to friends and family members would save lives. A similar percentage held negative beliefs about naloxone: 31.4% thought that distributing it will encourage people to use more opioid analgesics and 39.0% believed that preventing overdoses is ineffective because people will just continue to use and overdose again. Almost half of participants believed that naloxone should only be given by medical professionals (45.8%). 10.1371/journal.pone.0130050.t004 Table 4 Effects of messages on beliefs about naloxone distribution compared to the no-exposure control group. Message Exposure Belief a No-exposure control (n = 267) Factual information (n = 260) Factual information plus refutation (n = 266) Sympathetic narrative (n = 264) Sympathetic narrative plus factual information (n = 276) Sympathetic narrative plus factual information plus refutation (n = 265) Providing naloxone to first responders would save lives. (n = 1587) 60.0% (53.5 to 66.1) 73.7% (67.3 to 79.1) ** 76.1% (70.0 to 81.3) *** 77.0% (71.2 to 82.0) *** 88.4% (83.5 to 91.9) *** 83.5% (77.6 to 88.1) *** Providing naloxone to friends and family members would save lives (n = 1590) 35.9% (30.0 to 42.3) 55.8% (49.2 to 62.3) *** 58.6% (52.5 to 65.0) *** 58.8% (52.5 to 64.9) *** 73.9% (67.9 to 79.1) *** 72.9% (66.5 to 78.6) *** Distributing naloxone will encourage people to use even more opioid analgesics (n = 1591) 31.4% (25.6 to 37.7) 41.9% (35.6 to 48.5) * 28.1% (22.7 to 34.1) 31.6% (26.0 to 37.8) 34.8% (29.0 to 41.1) 26.9% (21.2 to 33.5) Preventing overdoses is ineffective because people will overdose again (n = 1589) 39.0% (33.0 to 45.4) 48.0% (41.5 to 54.5) 30.2% (24.7 to 36.4) * 31.0% (25.4 to 37.1) 32.0% (26.4 to 38.2) 22.2% (16.9 to 28.6) *** Naloxone should only be given by medical professionals (n = 1591) 45.8% (39.5 to 52.2) 34.5% (28.5 to 41.0) * 35.2% (29.3 to 41.6) * 40.2% (34.1 to 46.6) 25.8% (20.6 to 31.7) *** 24.3% (18.6 to 30.9) *** *P ≤ 0.05 compared to the no-exposure control group using logistic regression ** P ≤ 0.01 compared to the no-exposure control group using logistic regression *** P ≤ 0.001 compared to the no-exposure control group using logistic regression a Percent agreeing with beliefs calculated as percentage of sample responding 5, 6, or 7 on the seven point scale for each measure (i.e., somewhat agree, agree, or strongly agree) BODY.RESULTS.EFFECTS OF MESSAGES ON NALOXONE BELIEFS: In almost all cases, participants exposed to messages about naloxone had significantly different reported beliefs than those in the no-exposure control group (Table 4, Fig 2 and S2 Fig). Factual information alone led to more participants believing that providing naloxone to first responders would save lives (73.7% versus 60.0% in the no-exposure control group) and providing naloxone to friends and family members of people who use opioids would save lives (55.8% versus 35.9% in the no-exposure control group). Similarly, the sympathetic narrative alone led to more participants believing that providing naloxone to first responders (77.0% versus 60.0% in the no-exposure control group) and providing naloxone to friends and family members of people who use opioids (58.8% versus 35.9% in the no-exposure control group) would save lives. Participants in all message groups including factual information (i.e., alone or in combination with refutation and the sympathetic narrative) were less likely to believe that naloxone should only be given by medical professionals compared with participants in the no-exposure control group. Results from the sensitivity analysis using a belief scale were similar (S2 Appendix). 10.1371/journal.pone.0130050.g002Fig 2Effects of factual information, a sympathetic narrative, or both on beliefs about naloxone distribution. Provision of factual information alone led to higher agreement that distributing naloxone will encourage people to use more opioid analgesics compared with the no-exposure control group (41.9% versus 31.4%, respectively); this was not seen in the factual information plus refutation group (28.1% versus 31.4%). In addition, participants receiving factual information, compared with the no-exposure control group, were similarly likely to believe that preventing overdoses is ineffective because people will overdose again (48.0% versus 39.0%, respectively), but those receiving factual information plus refutation were less likely to believe this (30.2% versus 39.0%). Directly comparing the effects of adding the sympathetic narrative to factual information, using participants exposed to factual information alone as the referent group, we found that the combination led to a higher percentage of participants who believed that providing naloxone to first responders (OR: 2.7 [95% CI: 1.6 to 4.5]), and to friends and family members of people using opioid analgesics (OR: 2.2 (95% CI: 1.5 to 3.3]), would save lives. These participants were also less likely to agree that preventing overdoses is ineffective (OR: 0.5 [95% CI: 0.4 to 0.7]) and that naloxone should only be given by medical professionals (OR: 0.6 [95% CI: 0.4 to 0.99]). BODY.DISCUSSION: In a randomized, nationally-representative survey experiment, we found that public support for naloxone distribution policies was substantially higher after exposure to several different types of messages designed to educate participants about naloxone, refute common arguments against naloxone distribution, and evoke a sympathetic reaction toward people who use opioids. Our findings suggest that while most Americans do not currently support all naloxone distribution policies, there is an opportunity to improve public support through education and sympathetic portrayals of the population who stands to benefit from these policies. Compared to the control group, exposure to factual information alone led to higher support for providing naloxone to first responders as well as friends and family members of people who use opioids, suggesting that the general public has little knowledge of the safety and efficacy of naloxone. While the medication has been in routine clinical use for many years, it was not in widespread use in a "take-home" form to treat opioid overdoses until relatively recently. Furthermore, the concept of using a prescription medication to block an overdose of another prescription medication may be confusing; people may wonder if naloxone itself is a controlled substance with potential for recreational use, or think of it as analogous to methadone, an opioid agonist used to treat opioid use disorder, which also has the potential for overdose. Our finding that factual information alone can significantly increase support for naloxone distribution policies suggests that educating the public about naloxone's safety and efficacy will play an important role in garnering public support. While factual information alone increased support for several policies relative to no exposure, it also increased negative beliefs about naloxone. Participants receiving factual information only were significantly more likely to agree that distributing naloxone will encourage people to use even more opioid analgesics, and more likely to believe that preventing overdoses is ineffective, although this second comparison did not reach statistical significance. Learning more about how naloxone works potentially prompted some participants to think of ways it could be misused or be more receptive when those unintended consequences were presented to them. However, by adding a pre-emptive refutation message to factual information, those negative beliefs were mitigated. Future efforts to educate the public about the safety and efficacy of naloxone should pre-emptively address potential concerns about the unintended effects of naloxone distribution. The combination of a sympathetic narrative plus factual information appeared to be the most effective messaging approach for promoting support for naloxone distribution policies. Not only did this combination lead to significantly higher support for all policies relative to no message exposure, but it also led to higher support than that seen in participants receiving factual information alone. The percentage agreeing that providing naloxone to friends and family members would save lives approximately doubled, and there was no increase in negative beliefs about naloxone. Therefore, where possible, efforts to improve public support for overdose mortality prevention policies through education should also incorporate personal narratives to increase persuasiveness. Our finding that the sympathetic narrative was persuasive, both in isolation and over and above the provision of factual information about naloxone, has several potential explanations. Previous work has shown that messages evoking certain emotions (e.g., sympathy or sadness) for an individual representing a broader population can lead people to support policies that benefit that population [38]. This may be particularly important in the context of naloxone distribution to people who use opioids. As drug addiction is viewed as a moral failing by the majority of Americans, people who use drugs are often perceived very negatively [34,36]. Furthermore, harm reduction interventions (e.g., syringe exchange programs) have a long history of being highly controversial with the American public [47–49]. Messages evoking sympathetic reactions may overcome these negative attitudes toward people who use drugs and harm reduction interventions for them. In particular, results from the current study suggest that sympathetic portrayals of individuals whose lives are affected by others' drug addiction (e.g., the mother in our sympathetic narrative) may be successful in shaping support for harm reduction interventions by focusing on the dramatic consequences of overdose to non-users. BODY.DISCUSSION.LIMITATIONS: This study has several limitations. First, the exposure to the messages tested in in this Internet-based experiment may differ from the typical media environment where individuals are exposed to or select information about opioid analgesic overdoses and overdose prevention policies; our findings may not be representative of the impacts of real media content produced to convey these messages, however, themes of our messages were drawn from a convenience sample of actual news stories. Second, we tested only immediate responses to the messages, so the durability and stability of the assessed opinions is unclear. In addition, prior knowledge or experience with naloxone was not assessed and participants in the no-exposure control group, who were given no information about naloxone, may have drawn upon limited or no prior knowledge about the topic when answering survey questions. Third, we may not have identified or included other important messages which would change policy support and beliefs about naloxone in significant ways, such as news stories or video messages. Fourth, from the current analysis, we cannot exclude that differential effects of messages may be due to differences in content (e.g., information versus sympathetic narrative) as well as differences in other factors (e.g., word length). Finally, web-based studies are vulnerable to sampling biases. The GfK panel attempts to minimize such concerns by using address-based sampling to include households without a landline telephone. Furthermore, invitations to participate in surveys do not contain information about the content, so it is unlikely that participants chose whether or not to participate based on their interest in the topic. BODY.CONCLUSIONS: In summary, support for overdose mortality prevention policies was substantially higher among participants exposed to several different types of messages. Our findings that factual information alone can greatly improve support suggest that educating the public will be key in future efforts to widen naloxone distribution. Furthermore, education should be paired with pre-emptive refutation of concerns about potential unintended consequences of naloxone distribution. Finally, we found that a sympathetic narrative also improved public support, suggesting that sympathetic portrayals of people who use opioids through focusing on the loss faced by their family members is also effective in engendering support for naloxone distribution. Further research to determine which types of messages are optimal for subgroups of the population (e.g., by political ideology or education) will be useful in tailoring messages to increase persuasiveness. While a minority of Americans currently supports some of the overdose mortality prevention policies we studied, the results of our study provide public health officials and advocates with several effective tools to garner public support. BODY.SUPPORTING INFORMATION: S1 AppendixIntroductory text for the randomized survey experiment providing examples of opioid analgesics.(DOCX)Click here for additional data file. S2 AppendixDevelopment of a policy support and belief scale and effects of different message exposures on scaled outcomes.(DOCX)Click here for additional data file. S1 FigEffects of messages on support for naloxone distribution and other overdose mortality prevention policies.(TIF)Click here for additional data file. S2 FigEffects of messages on beliefs about naloxone distribution.(TIF)Click here for additional data file.

TITLE: Hepatitis B serological markers and plasma DNA concentrations ABSTRACT.OBJECTIVES:: To examine hepatitis B (HBV) serological markers and plasma DNA concentrations in a large group of untreated HBV/HIV-coinfected individuals in two sub-Saharan settings. ABSTRACT.DESIGN:: Baseline analysis of a randomized controlled trial. ABSTRACT.METHODS:: DART was a large trial of treatment monitoring practices in HIV-infected adults with advanced disease starting antiretroviral therapy at centres in Kampala or Entebbe, Uganda (n = 2317) and Harare, Zimbabwe (n = 999). HBV serological markers [antibody to HBV core antigen, HBV surface antigen (HBsAg), antibody to HBV surface antigen, HBV 'e' antigen (HBeAg), and antibody to hepatitis B 'e' antigen] and plasma HBV DNA viral load were measured retrospectively on stored baseline samples. Logistic regression was used to examine associations with baseline demographic and clinical factors. ABSTRACT.RESULTS:: The rate of HBsAg positivity was significantly higher in Zimbabwe than Uganda (12.2 vs. 7.7%, adjusted odds ratio = 1.54, P < 0.001) despite a similar prevalence of antibody to HBV core antigen (56.3 vs. 52.4%) in the two settings. Overall, HBsAg positivity was associated with male sex (adjusted odds ratio = 1.54, P < 0.001) but not with age, WHO disease stage, or CD4+ cell count. HBeAg was detected among 37% of HBsAg-positive patients, with higher rates among those with advanced WHO stage (P = 0.02). Also in HBsAg-positive patients, HBV DNA was undetectable in 21%, detectable but below the level of quantification in 14%, and quantifiable in 65%. A total of 96% of HBeAg-positive and 70% of HBeAg-negative patients had detectable HBV DNA; 92 and 28% of patients, respectively, had HBV DNA viral load more than 2000 IU/ml. ABSTRACT.CONCLUSION:: High rates of HBV coinfection were observed, highlighting the importance of ensuring that coinfected patients receive an antiretroviral regimen, whether first-line or not, that is active against both viruses. BODY.INTRODUCTION: In the era of antiretroviral treatment (ART), death rates from AIDS-related causes have declined dramatically in both resource-limited and resource-rich regions. In this context, liver disease has emerged as a major cause of death in HIV-infected individuals, although the absolute rates of liver-related mortality have declined [1–3]. Liver-related mortality is increased in those with viral hepatitis coinfection [4]. WHO HIV treatment guidelines state that it is important to determine the local prevalence of hepatitis B (HBV) to inform the decision whether to screen individuals for viral hepatitis, as recommended in resource-rich countries [5]. The use of ART to treat coinfected patients differs from treatment of HIV-monoinfected patients in a number of aspects, in particular ART regimens must have potent activity against both viruses, and treatment interruptions must be avoided because of the potential for liver flares [6–12]. We measured a comprehensive set of HBV serological markers and plasma HBV DNA viral load in archived baseline samples from over 3000 HIV-infected participants in the DART trial. This has allowed a detailed characterization of HBV/HIV coinfection in the regions from where participants were recruited, namely, Kampala/Entebbe, Uganda and Harare, Zimbabwe. BODY.METHODS: DART was a randomized open-label trial of monitoring practices in HIV-infected adult patients starting antiretroviral therapy, conducted at clinical centres in Uganda [Medical Research Council/Uganda Virus Research Institute (UVRI) Uganda Research Unit on AIDS, Entebbe (25 mi from Kampala); Joint Clinical Research Centre (JCRC), Kampala; and satellite Infectious Diseases Institute, Mulago, Kampala] and Zimbabwe (University of Zimbabwe, Harare). Patients were randomized to clinically driven monitoring only (CDM) or clinical monitoring and routine laboratory monitoring in the form of 12-weekly CD4+ and haematological/biochemical toxicity tests. Results were not returned for patients in the CDM arm unless specifically requested by the patient's doctor or if a grade 4 toxicity occurred. All participants started first-line ART with zidovudine and lamivudine and either tenofovir, nevirapine, or abacavir. Inclusion criteria were: age at least 18 years, CD4+ cell count less than 200 cells/μl, and naive to ART except for exposure for the prevention of mother-to-child transmission. Exclusion criteria were: likely to be unable to attend follow-up, likely to have poor compliance, acute infection including intense phase of tuberculosis treatment, malignancy requiring chemotherapy, laboratory test result indicative of contraindication to ART (including alanine transaminase greater than five times the upper limit of normal), pregnancy, and breastfeeding [13]. Plasma samples were stored at screening, enrolment, and each scheduled 3-monthly clinic visit. The current study describes the results of serological and virological tests for HBV that were performed retrospectively on the screening or enrolment sample for all participants, according to the algorithm in Fig. 1. All patients were tested for HBV surface antigen (HBsAg) and antibody to HBV core antigen (anti-HBc). Those with detectable anti-HBc without HBsAg were tested for antibody to HBV surface antigen (anti-HBs). Those with detectable HBsAg were tested for HBeAg, antibody to hepatitis B 'e' antigen (anti-HBe), and HBV DNA. Fig. 1Algorithm for hepatitis B serology testing.anti-HBc, antibody to hepatitis B core antigen; anti-HBe, antibody to hepatitis B 'e' antigen; anti-HBs, antibody to hepatitis B surface antigen; HBeAg, hepatitis B 'e' antigen; HBsAg, hepatitis B surface antigen; HBV DNA; hepatitis B DNA viral load. Serological and HBV DNA viral load assays were conducted locally, except for the testing of HBeAg, anti-HBe, and HBV DNA on UVRI samples, which were transported to and tested at JCRC. All sites used commercial serological assays [UVRI and Harare: Murex (Diasorin, Saluggia, Piedmont, Italy), based on an enzyme immunoassay method; JCRC: Roche Elecsys, an electrochemoluminescence assay (Roche Diagnostics Limited, Rotkreuz, Zug, Switzerland)]. The HBsAg assays were known to be unaffected by recognized HBsAg mutants; positive results were confirmed by a neutralization test. Anti-HBs results were classified as positive if the concentration was more than 10 mIU/ml. For the quantification of HBV DNA, JCRC used Roche Cobas Ampliprep/Cobas TaqMan (lower limit of detection 12 IU/ml, upper limit of quantification 110 × 106 IU/ml), whereas Harare used Abbott RealTime HBV after manually preparing samples using the mSample Preparation SystemDNA (lower limit of detection 10 IU/ml, upper limit of quantification 1 × 109 IU/ml). Due to low sample volumes, all samples at JCRC were diluted 1 : 4, giving a quantitative range of 48–440 × 106 IU/ml. Both JCRC and Harare participated in the United Kingdom National External Quality Assessment Service (UKNEQAS) scheme. Results from the three Uganda sites were broadly similar and have been combined in the analysis. BODY.METHODS.STATISTICAL METHODS: Multivariable logistic regression was used to examine associations between baseline factors and anti-HBc, HBsAg, HBeAg status, and HBV DNA viral load concentration (dichotomized using a cut-off of 2000 IU/ml [14,15]). P values for continuous and ordered variables (age, CD4+, and WHO stage) are presented from models fitting each variable as a continuous factor. Pairwise interactions between baseline factors (all possible combinations) were assessed by adding these to the model individually in addition to the main effects; in view of the large number of interaction terms examined, only those significant at P value less than 0.01 are reported. BODY.RESULTS: Selected baseline characteristics of the DART population are shown in Table 1 and have previously been reported in more detail [13]. There were 2317 participants from Uganda and 999 from Zimbabwe. Median age was 36 years, and women outnumbered men (ratio 1.9 : 1, P < 0.001). Reflecting the inclusion criteria, the population had advanced infection; median CD4+ cell count was 86 cells/μl, and 23% had previously been diagnosed with a WHO stage 4 illness. BODY.RESULTS.SEROLOGICAL FINDINGS: Data completeness was excellent, with only five (0.2%) participants not tested for anti-HBc and one (0.04%) not tested for HBsAg. The rate of anti-HBc positivity was similar in participants from the sites in Uganda (52%) and Zimbabwe (56%). Men were significantly more likely to test positive than women [adjusted odds ratio (aOR) 1.39, 95% confidence interval (CI) 1.19–1.61], although this difference was more marked in Zimbabwe (aOR 1.85, 95% CI 1.41–2.42) than Uganda (aOR 1.22, 95% CI 1.02–1.46) (P = 0.005, test for interaction). No association was observed with WHO stage or baseline CD4+ cell count. A total of 308 (9%) patients were found to be HBsAg-positive, 54 (18%) of whom were anti-HBc-negative, although most (n = 31) of them had other evidence of HBV infection in the form of detectable HBeAg, anti-HBe, or HBV DNA. A significantly higher rate (P = 0.001) of anti-HBc-negativity was observed in Zimbabwe (40/167; 24%) than in Uganda (14/140; 10%), and evidence of a higher rate at lower ages (P = 0.04, test for trend). No associations were observed for the other variables examined (sex, WHO stage, and CD4+ cell count). In contrast to the similar rates of anti-HBc positivity, HBsAg was detected much more frequently (aOR 2.99, 95% CI 2.35–3.81) in Zimbabwean patients (17%) than in Ugandan patients (6%). HBsAg positivity was significantly higher in men (aOR 1.54, 95% CI 1.20–1.97) but there was no association with age, WHO stage, or CD4+ cell count. Of 1505 patients who were anti-HBc-positive and HBsAg-negative, 962 (64%) were anti-HBs-positive, consistent with a resolved infection and natural immunity. A total of 280 (91%) patients who were HBsAg-positive had sufficient sample to allow further testing for HBeAg and anti-HBe. A total of 103 (37%) were HBeAg-positive and 127 (45%) were anti-HBe-positive; six (2%) patients had dual positive results and 56 (20%) dual negative results. HBeAg positivity was not associated with country or age, although there was a trend towards a higher prevalence in men (44%) than in women (31%) (P = 0.10) (Table 2). Rates of HBeAg positivity were higher in patients with more advanced HIV infection, as reflected by WHO stage (P = 0.02) and CD4+ cell count (P = 0.09). Further details of serological results are shown in Appendix 1. BODY.RESULTS.HEPATITIS B DNA VIRAL LOAD: Of 308 patients with a positive HBsAg result, 270 (88%) with available samples were tested for HBV DNA viral load. A total of 56 (21%) had undetectable DNA, 38 (14%) had DNA detectable but below the level of quantification, and 176 (65%) had a quantifiable level of DNA. The detection of HBV DNA viral load was strongly linked to HBeAg status, 96% (80/83) of HBeAg-positive and 70% (117/167) of HBeAg-negative participants having detectable levels (P < 0.001). Figure 2 shows the distribution of HBV DNA viral loads, by country and HBeAg status, in terms of the percentage of samples that exceed a given concentration. A vertical line is drawn at 2000 IU/ml, the threshold for initiating anti-HBV treatment according to some guidelines [14,15]. Overall, 92% of HBeAg-positive and 28% of HBeAg-negative participants had HBV DNA viral load more than 2000 IU/ml. As expected, HBV DNA levels were generally high among HBeAg-positive patients irrespective of clinical site, 53% having a value greater than 1 × 108 IU/ml. Values were spread more uniformly among HBeAg-negative patients and appeared on average to be lower in Zimbabwean than in Ugandan patients. Fig. 2Hepatitis B DNA viral load by hepatitis B 'e' antigen status. Table 3 shows the results of a multivariable logistic regression model predicting an outcome of HBV DNA viral load more than 2000 IU/ml. Models were fitted with and without a term for HBeAg status; these have different interpretations as both parameters are essentially measuring HBV replication. As suggested by Fig. 2, Zimbabwean patients were significantly less likely (P < 0.001) to have an HBV DNA level above this threshold, largely driven by differences among HBeAg-negative patients. The only other significant factor was WHO stage (in the model that did not adjust for HBeAg status), with a higher probability of having HBV DNA viral load more than 2000 IU/ml the more advanced the stage (P = 0.03). BODY.DISCUSSION: The description of the seroepidemiology of hepatitis B in HIV-infected adults in sub-Saharan Africa is largely limited to HBsAg and anti-HBc. In a systematic review of these markers, Barth et al.[16] reported an average HBsAg prevalence of 15%, but with a very wide range from 4 to 70%, and with variation occurring both between and within countries. The 6% HBsAg positivity rate found in DART participants from Kampala/Entebbe is somewhat lower than estimates from previous studies in this region of Uganda; the 17% rate in participants from Harare is somewhat higher than previous studies [17–25]. Notably, the overall prevalence of anti-HBc, with just over one-half of participants having evidence of exposure to the virus, was similar in the two countries. As vertical transmission or infection in the first few years of life is the strongest determinant of developing chronic infection, this suggests that the proportion infected early in life is higher in Zimbabwe than in Uganda. We found a slight increase in the prevalence of anti-HBc with increasing age, which may indicate continuing infection during adulthood but may also be a cohort effect, with historically declining transmission. HBsAg was detectable despite undetectable anti-HBc in 54 (1.6%) study participants. The higher rate in Zimbabwe could be due to biological differences between the populations or the use of different serological assays. The prevalence of this atypical pattern has been described to range between 4 and 56% of those with detectable HBsAg [17,26], and in differing situations including in neonates, in immunosuppression, and in the presence of core gene mutations [26–29]. In HIV-positive individuals, it is associated with a low CD4+ cell count, sometimes with development of an anti-HBc response on starting ART [30]. As an anti-HBc test is sometimes used to screen patients prior to an HBsAg test, this testing strategy may fail to identify some HBsAg-positive patients [31]. A total of 543 participants, 30.0% of those with evidence of HBV exposure, had isolated anti-HBc. Similar rates (32–42%) have been found in previous studies in Uganda and elsewhere in sub-Saharan Africa [32–35]. This pattern may be due to false-positive anti-HBc test results, rare in a high prevalence population such as this, or be transient and occur during the resolution phase of acute HBV. Persistent isolated anti-HBc may also be due to occult HBV infection (with low-level detectable HBV DNA viral load) or loss of anti-HBs with time or immunosuppression in patients who have resolved infection. Repeat serology and HBV DNA viral load testing would help to determine more accurately the status of the 543 patients with isolated anti-HBc, but was not available in this study. The major novel contribution from our study in an HIV-positive population in Africa is extensive data on HBeAg and HBV DNA viral load, the most powerful prognostic markers for disease progression and viral transmission. Previous studies are either based on small sample sizes or do not distinguish HIV-uninfected and HBV/HIV-coinfected individuals. A previous study of mostly HIV-negative, HBsAg-seropositive inpatients in Kampala found 27% HBeAg seropositive [22]. An earlier study of inpatients in the same hospital found six (28.1%) of 23 HIV-positive and three (17.6%) of 17 HIV-negative patients to be HBeAg seropositive [18]. A small study of HIV-infected pregnant women in Uganda and Rwanda found that three (33%) of nine with detectable HBsAg were HBeAg seropositive [19]. In Zimbabwe, rates of HBeAg seropositivity ranged from 3.3% in pregnant women in Harare [36] to 24.5% [37] in a national survey, but neither study tested for HIV. In HIV/HBV-coinfected Zimbabwean patients recruited to a randomized controlled trial, 54.2% (13 of 24) were HBeAg seropositive [25]. In the DART population, we found that approximately one-third of HBsAg-positive patients were HBeAg positive. As expected, HBeAg status was intimately linked to HBV DNA viral load, with very high levels observed in HBeAg-positive patients. Nonetheless, 28% of HBeAg-negative patients had a viral load greater than 2000 IU/ml, the threshold for considering anti-HBV treatment in guidelines [14,15]. In the small study of HBV/HIV-coinfected pregnant women in Uganda and Rwanda, three of five HBeAg-negative patients had detectable HBV DNA with a mean viral load of 1700 IU/ml [19]. In the randomized controlled trial cited above [25], the 24 Zimbabwean patients were included in a larger cohort (n = 115) in which at least 28% of HBeAg-negative participants had HBV DNA greater than 2000 IU/ml [10]. Among HBeAg-positive patients, HBV DNA was detected more frequently, and the distribution of viral load values was higher in Uganda than in Zimbabwe. The use of different viral load assays is one possible explanation for this finding, although both laboratories participated in the same external quality control scheme. Immunosuppression associated with HIV coinfection can result in reactivation of HBV infection, with reappearance of HBsAg or HBeAg, or a reduction in the rate of loss of either marker over time. Without an HIV-negative comparator population, we were not able to examine this; however, we were able to assess the influence of the degree of immunosuppression as measured by WHO clinical stage and CD4+ cell count at study entry. No clear associations were observed for HBsAg status, but HBeAg positivity was markedly higher in those with a more advanced WHO stage of disease, and there was a consistent, albeit nonsignificant, trend with CD4+ cell count. The effect of WHO stage was mirrored in an analysis of the proportion of participants with HBV DNA levels greater than 2000 IU/ml. Reactivation of HBV infection could explain the higher liver-related mortality that has been observed in HBV/HIV-coinfected individuals [4]. An association between advanced disease (in this case low CD4+ cell count) and both HBeAg and HBsAg status was noted previously in a study in Nigeria, although these investigators suggested causation to be acting in the other direction, namely, active HBV infection lowering CD4+ cell count [38]. The prevalence of anti-HBc was significantly higher in male participants than in female participants, particularly in Zimbabwe. An even more pronounced sex difference was observed for HBsAg, consistent with other studies that have shown that men are less likely to clear HBV and progress to chronic infection, either when infected in childhood or as adults [39–42]. There was also a nonsignificant trend of a higher rate of HBeAg positivity among male participants, although no evidence of a sex difference in HBV DNA concentrations exists. The strengths of our study are the large sample size, the very high rate of sample retrieval (close to 100%), detailed clinical and demographic data, the comprehensive range of virological markers tested, and that all the laboratories were participating in the UKNEQAS quality assurance programme. The main limitation is the testing of participants at a single time point, precluding the estimation of HBV incidence and the ability to determine with certainty that all the HBsAg positivity was due to chronic HBV infection or whether there may have been some acute infections that may resolve. It is unlikely that acute infection contributes substantially to HBsAg prevalence as most transmission in sub-Saharan Africa occurs in childhood. Another limitation is that we did not perform HBV DNA assays on the surprisingly large number of patients (n = 543) who had isolated anti-HBc. This serological pattern may represent a false-positive, resolved and cleared infection, or chronic infection with a low rate of viral replication (occult HBV infection). In a previous small study in Uganda, 15% (seven of 48) HIV-positive patients with negative HBsAg had detectable HBV DNA [22]. The clinical implications of occult HBV infection are unclear, but it is generally accepted that individuals with detectable plasma HBV DNA may be at risk of HBV reactivation and inflammatory liver flares [43]. Finally, DART was limited to patients with a CD4+ cell count less than 200 cells/μl and thus did not include those with less-advanced HIV infection. This may have limited our ability to identify associations with this key marker of immunosuppression. In conclusion, high rates of active HBV infection were observed in both geographical settings in Africa, highlighting the importance of considering HBV coinfection in patients receiving antiretroviral drugs, regardless of whether this is their first or subsequent regimen, and using agents that are active against both viruses. Further analyses of longitudinal data in DART are ongoing, including the impact of chronic HBV infection on mortality and whether coinfected patients who received lamivudine without tenofovir had less favourable virological outcomes compared with those who received both drugs. BODY.ACKNOWLEDGEMENTS: We thank all the patients and staff from all the centres participating in the DART trial. DART was funded by the UK Medical Research Council (grant number G0600344), the UK Department for International Development (DFID), and the Rockefeller Foundation. GlaxoSmithKline/ViiV Healthcare, Gilead, Boehringer Ingelheim, and AbbVie Inc. donated drugs for DART. Gilead Sciences funded the HBV immunological and virological assays. H.P. was funded by a UK Medical Research Council Clinical Research Training Fellowship. Authors' contributions: H.P. – designed and coordinated study, carried out analysis, wrote first draft, and revised subsequent versions; D.D. – designed and coordinated study, advised on analysis, cowrote first draft, and revised subsequent versions; T.Z. and T.V. – carried out laboratory testing, commented on drafts; M.C. – carried out laboratory testing; C.K., P.M., and J.H. – site PI for main trial, commented on drafts; M.S. – coordinated study, commented on drafts; C.G. and D.P. – designed study, commented on drafts; P.K. – commented on drafts; R.G. – designed and coordinated study, commented on drafts. Justification of the number of contributors greater than 10: DART was a very large international study with a large study group. DART Virology Group: P. Kaleebu (Co-Chair), D. Pillay (Co-Chair), P. Awio, M. Chirara∗, D. Dunn, D.M. Gibb, C. Gilks, R. Goodall, A. Kapaata, M. Katuramur, F. Lyagoba, B. Magambo, K. Mataruka, L. Mugarura, T. Musunga, M. Nabankkema, J. Nkalubo, P. Nkurunziza, C. Parry, V. Robertson, M. Spyer, D. Mulima, D.E. Williams, I. Nankya, S. Nassimbwa, E. Ndashimye, E. Nabulime, M. Phiri, K. Mutasa, and S. Mukasa. MRC/UVRI Uganda Research Unit on AIDS, Entebbe, Uganda: H. Grosskurth, P. Munderi, G. Kabuye, D. Nsibambi, R. Kasirye, E. Zalwango, M. Nakazibwe, B. Kikaire, G. Nassuna, R. Massa, K. Fadhiru, M. Namyalo, A. Zalwango, L. Generous, P. Khauka, N. Rutikarayo, W. Nakahima, A. Mugisha, J. Todd, J. Levin, S. Muyingo, A. Ruberantwari, P. Kaleebu, D. Yirrell, N. Ndembi, F. Lyagoba, P. Hughes, M. Aber, A. Medina Lara, S. Foster, J. Amurwon, and B. Nyanzi Wakholi. Joint Clinical Research Centre, Kampala, Uganda: P. Mugyenyi, C. Kityo, F. Ssali, D. Tumukunde, T. Otim, J. Kabanda, H. Musana, J. Akao, H. Kyomugisha, A. Byamukama, J. Sabiiti, J. Komugyena, P. Wavamunno, S. Mukiibi, A. Drasiku, R. Byaruhanga, O. Labeja, P. Katundu, S. Tugume, P. Awio, A. Namazzi, G.T. Bakeinyaga, H. Katabira, D. Abaine, J. Tukamushaba, W. Anywar, W. Ojiambo, E. Angweng, S. Murungi, W. Haguma, S. Atwiine, and J. Kigozi. University of Zimbabwe, Harare, Zimbabwe: A. Latif, J. Hakim, V. Robertson, A. Reid, E. Chidziva, R. Bulaya-Tembo, G. Musoro, F. Taziwa, C. Chimbetete, L. Chakonza, A. Mawora, C. Muvirimi, G. Tinago, P. Svovanapasis, M. Simango, O. Chirema, J. Machingura, S. Mutsai, M. Phiri, T. Bafana, M. Chirara, L. Muchabaiwa, and M. Muzambi. Infectious Diseases Institute (formerly the Academic Alliance) Makerere University, Mulago, Uganda: E. Katabira, A. Ronald, A. Kambungu, F. Lutwama, A. Nanfuka, J. Walusimbi, E. Nabankema, R. Nalumenya, T. Namuli, R. Kulume, I. Namata, L. Nyachwo, A. Florence, A. Kusiima, E. Lubwama, R. Nairuba, F. Oketta, E. Buluma, R. Waita, H. Ojiambo, F. Sadik, J. Wanyama, and P. Nabongo. The AIDS Support Organisation (TASO), Uganda: R. Ochai and D. Muhweezi. Imperial College, London, UK: C. Gilks, K. Boocock, C. Puddephatt, D. Winogron, and J. Bohannon. MRC Clinical Trials Unit at UCL, London, UK: J. Darbyshire, D.M. Gibb, A. Burke, D. Bray, A. Babiker, A.S. Walker, H. Wilkes, M. Rauchenberger, S. Sheehan, L. Peto, K. Taylor, M. Spyer, A. Ferrier, B. Naidoo, D. Dunn, and R. Goodall. Independent DART Trial Monitors: R. Nanfuka and C. Mufuka-Kapuya. Trial Steering Committee: I. Weller (Chair), A. Babiker (Trial Statistician), S. Bahendeka, M. Bassett, A. Chogo Wapakhabulo, J. Darbyshire, B. Gazzard, C. Gilks, H. Grosskurth, J. Hakim, A. Latif, C. Mapuchere, O. Mugurungi, P. Mugyenyi; Observers: C. Burke, M. Distel, S. Jones, E. Loeliger, P. Naidoo, C. Newland, G. Pearce, S. Rahim, J. Rooney, M. Smith, W. Snowden, J.-M. Steens, and M. Ait-Khaled. Data and Safety Monitoring Committee: A. Breckenridge (Chair), A. McLaren (Chair-deceased), C. Hill, J. Matenga, A. Pozniak, and D. Serwadda. Endpoint Review Committee: T. Peto (Chair), A. Palfreeman, M. Borok, and E. Katabira. BODY.ACKNOWLEDGEMENTS.CONFLICTS OF INTEREST: There are no conflicts of interest. BODY.SUPPLEMENTARY MATERIAL: Supplemental Digital Content

TITLE: A randomized trial of fish oil omega-3 fatty acids on arterial health, inflammation, and metabolic syndrome in a young healthy population ABSTRACT.BACKGROUND: Long chain omega-3 fatty acids from fish oils (O3) are known to have beneficial effects on a number of vascular risk factors in at-risk populations. The effects of a highly bioavailable emulsified preparation on an overweight young adult population are less well known. ABSTRACT.METHODS: Young adults, age 18–30, with body mass indices (BMIs) greater than 23 (average = 28.1) were administered 1.7 g of O3 per day (N = 30) or safflower oil placebo (N = 27) in an emulsified preparation (Coromega, Inc.) for 4 weeks in a double-blind randomized design. Blood was drawn and anthropometric measurements taken before and after dosing. Hemodynamic measures (central pulse wave velocity, augmentation index, and aortic systolic blood pressure), inflammatory cytokines (IL-6, IL-8, IL-10, and tumor necrosis factor-α), red blood cell and plasma phospholipid fatty acid profiles, fasting serum lipids, glucose, and C-reactive protein were measured. ABSTRACT.RESULTS: Red cell and plasma phospholipid eicosapentaenoic acid and docosahexaenoic acid concentrations increased over the four weeks of dosing in the O3 group. Dosing with O3 did not affect central pulse wave velocity, augmentation index, or aortic systolic blood pressure. None of the five American Heart Association metabolic syndrome components improved over the dosing period. None of the inflammatory cytokines, C-reactive protein, or lipids (total or LDL cholesterol) improved over the dosing period. ABSTRACT.CONCLUSIONS: No salutary effects of O3 were observed in hemodynamic, metabolic syndrome criteria or inflammatory markers as a result of this relatively short period of administration in this relatively overweight, but healthy young adult cohort. BODY.BACKGROUND: Metabolic syndrome has become highly prevalent in the U.S. and has a strong impact on the development of future vascular ailments including Type II diabetes mellitus and cardiovascular diseases. This increase can be partially attributed to recent changes in the American diet that negatively affect body weight and arterial health [1]. Arterial stiffness is associated with metabolic syndrome and is a predictor of cardiovascular events [2]. Pulse wave velocity (PWV) and augmentation index (AIx) are measurements of arterial distensibility [3]. Reducing dietary saturated fats and increasing omega-3 polyunsaturated fats, especially from fish (O3), have long been known to improve vascular health and may improve measures of arterial stiffness [4]. Dangardt et al. reported supplementation with O3s improved vascular function and lowered the severity of inflammation among the obese [5]. However, Mackay, et al. found fish oil supplementation among those with or at risk of heart disease and receiving aspirin and statin therapy had no effect on pulse wave velocity [6]. O3s have also been found to improve obesity-induced metabolic syndrome through regulating chronic inflammation [7]. These include C-reactive protein (CRP), interleukin-6 (IL-6), and tumor-necrosis factor-α (TNF-α) [8]. While previous researchers have focused their studies on disease risk factors in older at-risk populations [9,10], the present study sought to investigate the effects of O3 supplementation in a relatively overweight college-aged population. The pathologies of vascular diseases have been observed and recorded in the very young. Earlier intervention in those at early risk may show greater promise in longer-term benefits. Researchers have found that younger populations have responded favorably to O3 dosing during weight loss in reducing triglycerides, leptin, insulin, insulin resistance, and blood pressure and increasing ghrelin concentrations [11-14]. In addition, younger adults tend to consume relatively low amounts of fish and have relatively low levels of O3s in blood and tissues [15]. This might make this group especially responsive to the beneficial effects of even low doses of O3. Others have also found high PWV and Aix values among obese adolescents [16]. Thus, this study investigated whether O3s improved arterial health and inflammatory responses, both being indicators of risk for metabolic syndrome and ultimately, heart disease, diabetes, and stroke, in a healthy young relatively overweight population. BODY.METHODS.SUBJECTS: Healthy 18–30 year old men and women were recruited from the Appalachian State University student population. 430 responded. Respondents were sent a survey to determine inclusion and exclusion criteria, including self-reported height and weight and weekly exercise engagement. Of those initial surveys, 343 were returned. Sixty subjects were invited to participate in the study based on the following inclusion criteria: body mass index (BMI) >23 and engagement in regular exercise of no more than 3 times per week. Subjects were also not taking any cardiovascular medications or fish oil supplements, not allergic to fish, or with a history of diabetes, heart disease or a stroke, and did not consume fish more than twice per week. The research protocol was approved by the Institutional Review Board of Appalachian State University. BODY.METHODS.STUDY DESIGN: The initial treatment and control groups consisted of 30 and 27 participants, respectively. Three subjects who were invited to enter the study failed to attend the first clinical visit. Randomization was performed from a random digits table. The subjects recruited for this four-week, double-blind study completed two visits to the clinic. At the first clinical visit, anthropometric measurements, including height, weight, and waist circumference, and seated blood pressure were measured by a single trained technician. Hemodynamic testing included central PWV and AIx, which is further used to determine aortic pressure [17]. Hemodynamic testing was performed by 3 different trained technicians. A fasting blood sample was drawn for blood lipids, red blood cell and plasma phospholipid fatty acid profiles, high sensitivity C-reactive protein (hsCRP), blood glucose, and inflammatory cytokines. A Health History Questionnaire was completed at home and returned within the first week. Participants were requested to comply with their reported usual exercise habits of three or less times per week during the study period. At the conclusion of the first clinical visit, subjects were supplied with four weeks' worth of O3 or placebo oil single-dose packets. Treatment consisted of 4 weeks of supplementation with O3, 350 mg eicosapentaenoic acid (EPA) and 230 mg docosahexaenoic acid (DHA) per single-dose packet, while the placebo was 1.0 g of safflower oil per packet. The oils were emulsified products (Coromega, Inc., Vista, CA) provided in three premeasured packets; one packet to be opened and consumed at breakfast, two packets at dinner. Protocol compliance was monitored with a check sheet that participants kept to record the supplements they consumed. A second clinical visit occurred at the end of the intervention period of four weeks and included a second round of clinical measures, blood draws and hemodynamic testing as before. BODY.METHODS.MEASUREMENTS: Height and weight were determined using a Healthometer (Jarden Corp, Rye, NY) stadiometer and scale. Waist circumference was measured manually with a tape measure. Blood pressure was measured using a manual sphygmometer according to AHA guidelines. Blood lipids, glucose, and CRP were measured at the local hospital laboratory. Hemodynamic and vascular testing was performed with a manual blood pressure cuff and sphygmomanometer and the Sphygmacor Cardiovascular Management System. Applanation tonometry (SphygmoCor, Inc., Sydney, Australia) was used to derive the range of central arterial indices, including PWV and AIx [17]. The same trained technician preformed each measurement and our laboratory technicians have an intra-class correlation coefficient of 0.96 with a coefficient of variation (%CV) of <4.0%. Serum lipids, glucose, and hsCRP were measured on a Dimensions RXL instrument from Siemens, Inc. Total cholesterol was measured by polychromatic endpoint technique using oxidase, peroxidase and esterase with a%CV of 1.82%. HDL cholesterol was measured by the accelerator selective detection method (direct measure polymer-polyanion) with a 0.92%CV. Triglycerides were measured by enzyme immunoassay with bichromatic endpoint with 3.60%CV. LDL cholesterol was measured by direct measure enzyme immunoassay with bichromatic endpoint with 2.72%CV. Glucose was measured by hexokinase with bichromatic endpoint with 3.50%CV. Finally, hsCRP was measured with colorimetric immunoassay with bichromatic endpoint with 2.1%CV. Levels of inflammation protein targets were measured using the Meso Scale Discovery® Multi-Spot® Assay System. In this multiplex electrochemiluminescent ELISA, IL-6, IL-8, IL-10, and TNF-α concentrations were detected on spatially distinct spots in single wells on 96-well plates. Sample analyses were performed in duplicate. Intra-assay coefficient of variability between assay plates was 11% for IL-6, 5% for IL-8, 8% for IL-10, and 7% for TNF-α. Fatty acid analysis was performed essentially according to the method of Lands, et al [18]. Briefly, sample lipids were extracted by the method of Bligh and Dyer [19]. Lipid fractions were separated on thin layer chromatography. Appropriate samples were then transesterified with boron trifluoride and extracted. Methyl esters were separated and quantified with a Shimadzu capillary gas chromatograph with flame-ionization detection. Authentic standards and internal standards were used. The %CVs for the long-chain omega-3 fatty acids were under 5%. BODY.METHODS.STATISTICAL ANALYSIS: Repeated measures general linear models were used to determine treatment effects. Chi Square and Student t-tests were used to determine differences between treatment groups at baseline. Analysis was performed with SPSS v.18 (SPSS, Inc. Chicago, Illinois). A power calculation was made to estimate group size a priori. A total of 30 subjects were required to give us adequate statistical power at a p < 0.05 for the outcome variables of PWV and AIx. Based on existing data from our laboratory, the estimated sample size of 30 subjects gives us an effect size of 0.92 and 0.88, respectively, with an alpha set at 0.05. Test-retest reliabilities in our laboratory for these 2 dependent variables are 0.98 and 0.97, respectively. BODY.RESULTS: Of the 57 men and women who entered the study, 51 completed the intervention. Six participants did not return for the second clinical visit, four from the O3 group and two from the placebo group. Of those that completed the study, 41 returned the compliance check sheets, 19 from the placebo group and 22 from the fish oil group. Of those, 100% of the placebo participants consumed greater than 85% of their supplements while 86% of the fish oil participants consumed greater than 85% of their supplements. Anthropometric measurements and blood samples were collected during the initial clinical visit. Table 1 summarizes baseline information collected. Subjects chosen were at risk of metabolic syndrome with blood pressure measurements and waist circumferences slightly higher than normal, particularly for women. There were no statistically significant differences in baseline measurements between the treatment groups. Table 1 Baseline information of study subjects (percent or mean ± standard deviation)* Variable Placebo group Omega-3 group N 27 30 Age (years) 20.4 ± 2.1 21.4 ± 2.9 Female 38% 48% Body mass index (kg/m 2 ) 27.9 ± 2.9 28.5 ± 3.3 Systolic blood pressure (mmHg) 125 ± 7 124.5 ± 7 Waist circumference, females (cm) 86 ± 10 90 ± 11 Waist circumference, males (cm) 92 ± 8 96 ± 6 Triglycerides (mmol/L) 1.01 ± 0.56 1.01 ± 0.52 HDL-cholesterol (mmol/L) 1.22 ± 0.32 1.20 ± 0.26 Serum glucose (mmol/L) 4.7 ± 0.4 4.7 ± 0.4 Activity level (number times exercise per week) 3.0 ± 0.9 3.0 ± 0.9 Current Smokers 25% 8% * None of the groups were statistically significantly different at p < 0.05. Measurements were also taken four weeks later, after O3 supplementation. Figure 1 shows the effect of O3 dosing for four weeks on plasma phospholipid EPA and DHA. Plasma levels increased significantly (p < 0.001). Figure 1Effect of fish oil supplementation on plasma phospholipid combined EPA and DHA levels as percent of total fatty acids before and after treatment. Plasma and red blood cell phospholipid fatty acid outcomes are displayed in Table 2. Significant increases are noted for both EPA and DHA and their combination in both samples. In the plasma the combined EPA and DHA increased about 80% and in the red blood cells the combined fatty acids increased about half as much. Table 2 Effect of fish oil supplementation on plasma and red blood cell phospholipid fatty acids* Markers Placebo Omega-3 Treatment difference S.E. of difference P-value of difference Before After Before After Plasma PL% EPA 0.332 0.400 0.329 1.225 +0.832 0.170 p < 0.001 Plasma PL% DHA 1.623 1.370 1.618 2.157 +0.762 0.281 p = 0.008 Plasma PL% EPA&DHA 1.95 1.77 1.95 3.38 +1.61 0.408 p < 0.001 RBC% EPA 0.314 0.373 0.366 1.026 +0.606 0.108 p < 0.001 RBC% DHA 2.930 3.082 3.169 4.169 +0.708 0.245 p = 0.019 RBC% EPA&DHA 3.244 3.455 3.535 5.195 +1.314 0.298 p = 0.002 * Repeated measure general linear models. PL = phospholipid, EPA = eicosapentaenoic acid, DHA = docosahexaenoic acid. The effects of supplementation on components of the metabolic syndrome, plasma lipids, vascular measures, and cytokines are shown in Table 3. The daily treatment dose of 1.7 g of O3 for four weeks had no effect on any of these measures compared to the safflower placebo. Not shown are BMI, waist, total cholesterol, serum glucose, IL-8, and IL-10, all of which also showed no effect. Table 3 Effect of fish oil supplementation on arterial measures* Markers Placebo Omega-3 Treatment difference S.E. of difference P-value of difference Before After Before After Aortic systolic blood pressure (mmHg) 112.1 110.0 110.8 111.1 +2.54 1.70 0.91 Central PWV (m/s) 7.44 6.61 7.49 7.43 +0.76 0.70 0.26 Augmentation Index (%) 10.8 7.4 10.9 11.6 +4.00 2.73 0.47 SBP (mmHg) 125.7 124.5 124.5 125.1 +1.9 1.7 0.26 LDL cholesterol (mmol/L) 2.41 2.39 2.47 2.48 -0.01 0.15 0.81 Triglycerides (mmol/L) 0.88 0.99 0.91 0.91 -0.099 0.145 0.83 HDL-cholesterol (mmol/L) 1.225 1.164 1.204 1.189 +0.004 0.050 0.74 TNF-α (pg/mL) 4.90 4.52 5.31 5.70 +0.14 0.08 0.75 IL-6 (pg/mL) 0.87 0.77 0.99 1.12 +0.21 0.19 0.51 CRP (nmol/L) 20.7 21.8 17.3 18.7 +0.12 0.34 0.53 * Repeated measure general linear models. PWV = Central pulse wave velocity, BMI = Body Mass Index, SBP = Systolic Blood Pressure. TNF-α = Tumor Necrosis Factor-α, IL-6 = Interleukin-6, CRP = C - reactive protein. Triglycerides, TNF-α, IL-6, and CRP are log transformed; means expressed as geometric means; differences are expressed as log values. BODY.DISCUSSION: In this study we investigated the effect of O3 on relatively overweight but healthy young adults. We found no effect of 4 weeks of dosing with 1.7 grams per day on arterial hemodynamic measures, components of the metabolic syndrome, serum lipids, or measures of chronic inflammation. Yet, we were able to verify a change in O3 status in serum and red cell phospholipids with O3 dosing. Emulsifying fish oils can enhance digestion and absorption of the fatty acids. Raatz, et al. investigated emulsified fish oil absorption compared with capsular triglyceride fish oil supplements in humans throughout a 48-hour observation period. A single dose (350 mg EPA and 230 mg DHA) of the emulsified product resulted in enhanced absorption of total O3 compared with the capsular supplement. Although we gave a relatively low dose, Raatz, et al. have shown that this material is absorbed more quickly and maybe more completely than fish oil in tablets [20]. Figure 1 shows that the O3 treatment increased plasma O3 EPA and DHA, despite four apparent non responders in the treatment group and two with increased EPA and DHA in the placebo group. Arterial stiffness is associated with metabolic syndrome [2]. PWV and pulse pressure are measures positively associated with aortic stiffening, also measured by AIx [3,21]. Sjoberg, et al. introduced 2 g, 4 g, and 6 g of fish oil supplementation per day into the diets of overweight or obese adults for 12 weeks. Improvement in arterial distensibility, as measured by PWV, was only found to be significant only at the highest dose of 6 g of fish oil per day [22]. Chong, et al. reported a significant improvement in PWV and AIx, in healthy adults immediately after a long chain O3 PUFA-rich meal containing 4.7 g of DHA and EPA [4]. However, Sanders, et al. recently found that 1.8 g of EPA and DHA daily over 12 months did not improve arterial stiffness among slightly overweight but relatively healthy middle aged subjects in England [23]. Thus, using a comparable dose over a greater duration than in our study within an older age group yielded similar outcomes. A number of studies have been conducted on the association between O3 intake and the development of the metabolic syndrome. The present study found that fish oils did not have an effect on components of the metabolic syndrome in overweight young adults. Pederson, et al. found that supplementation with 1.5 g of O3 per day for 16 weeks significantly lowered systolic blood pressure and raised HDL cholesterol in overweight adolescents [24]. Also, supplementation with O3 over a 12-week period significantly lowered serum glucose levels [25]. Inflammation is also recognized as having a significant relationship with metabolic syndrome [26]. Dietary patterns poor in O3 may cause an excessive production of pro-inflammatory cytokines and CRP, while causing a lower production of anti-inflammatory cytokines, all recognized as contributing to the inflammation associated with metabolic syndrome and cardiovascular events [26,27]. Dangardt, et al. executed an intervention of 1.2 g of O3 supplements per day on obese adolescents for 3 months having an average BMI of 33.8, compared to our group average BMI of 28.1. Results showed a significant decrease in TNF-α and IL-6 levels, but no significant change in the serum levels of CRP, IL-8 or IL-10 [5]. Low CRP levels have also been observed in Yup'ik Eskimos, a population who have mean daily intakes of DHA and EPA ranging from 2.4 to 3.7 g. In this population, CRP blood levels were inversely related to the intake of fish oils; however, there was no relationship found with IL-6 levels and fish oil intake [28]. Our results, in addition to those found in past studies, may further demonstrate the need for a longer intervention period, a higher treatment dose and an at-risk population selection for treatment to observe the desired results in inflammatory markers. Overall, our present investigation found little effect of emulsified fish oil on components of the metabolic syndrome, inflammatory cytokines, or hemodynamic measures of arterial health. Other studies suggest that higher doses of fish oil coupled with a longer intervention period executed in a more unhealthy population may be needed to manifest positive and significant results from O3 intervention. Strengths of our study include the bioavailability of the emulsified supplement, clear evidence for incorporation of O3 doses into blood phospholipids, the young age of the participants, and the multiplicity of the endpoints, specifically the hemodynamic markers and the inflammatory cytokines. However, some limitations to the study are apparent. These include the short length in time of the dosing and the relatively low dose compared to other more recent studies. BODY.CONCLUSIONS: The administration of a highly bioavailable emulsified preparation of fish oil O3 measurably increased plasma and red blood cells EPA and DHA in four weeks. However, no beneficial changes were observed in the markers that were measured for hemodynamic, metabolic syndrome, or inflammatory effects as a result in this relatively healthy overweight young adult cohort. More at-risk populations may benefit more from this intervention. BODY.ABBREVIATIONS: CRP: C-reactive protein; DHA: Docosahexaenoic acid; EPA: Eicosapentaenoic acid; IL-6: Interleukin-6; IL-8: Interleukin-8; IL-10: Interleukin-10; O3: Fish oil omega-3 fatty acids; TNF-α: Tumor necrosis factor-alpha. BODY.COMPETING INTERESTS: Martin Root received funding for this research from the Dyson Foundation and the supplements from Coromega, Inc. The other authors declare that they have no competing interests. BODY.AUTHORS’ CONTRIBUTIONS: MR was the Principal Investigator. He directed the study, the data analysis and the drafting of the manuscript. He finalized and submitted the manuscript. SC directed the arterial function testing and data interpretation and hosted study site. KZ conducted the inflammatory marker analysis and data interpretation. KW wrote the initial manuscript and performed the data analysis. MM managed the trial, recruiting subjects, scheduling both subjects and researchers and coordinating data collection. All authors reviewed and contributed to the final version of the manuscript.

TITLE: Comparison of paracetamol and fentanyl for pain relief during and after suction termination ABSTRACT.OBJECTIVES:: To compare the combination of paracetamol (20 mg/kg) and propofol to fentanyl (1 μg/kg) and propofol in terms of providing adequate analgesia and a comparable recovery profile in suction termination procedures. ABSTRACT.METHODS:: This is a prospective, randomized clinical study in which we obtained informed consents from 146 women (fentanyl group: 76 [52.1%], paracetamol group: 70 [47.9%]) who were scheduled for suction curettage at the Istanbul Kanuni Sultan Suleyman Education and Training Hospital, Istanbul, Turkey in January 2015. Patients were randomly allocated into a fentanyl group or a paracetamol group. Visual analogue scores, modified Aldrete scores, and hemodynamic parameters were recorded during and after the surgical procedure. A record was also maintained of any adverse events. ABSTRACT.RESULTS:: When the modified Aldrete scores at 60 minutes, systolic pressures at 0 minutes, oxygen saturation at 10, 15, 20 minutes, diastolic blood pressure at 10, 15, 20 minutes, heart rates, and visual analogue scores were compared, there was no significant difference between groups (p>0.05). In the fentanyl group, systolic blood pressures at 5, 10, 15, 20 minutes and diastolic blood pressure at 5 minutes and oxygen saturation at 5 minutes were significantly lower (p<0.05). ABSTRACT.CONCLUSION:: Our study demonstrates that the analgesic properties and recovery profiles of intravenous paracetamol is as effective as fentanyl when used in suction termination procedures. Further larger studies are still required. BODY: Suction curettage is a standard surgical technique and is commonly used for first-trimester pregnancy terminations. One of the important features of this procedure is the shortness of its duration. Patients are typically day cases and are discharged on the same day of the surgery. There can usually be serious pain involved in the procedure;1,2 however, patients usually experience pain during gynecologic procedures that involve the uterine cavity.3 Besides ensuring the use of an effective analgesic, another important factor in brief surgical interventions such as suction termination is having a fast recovery time. Paracetamol (acetaminophen; N-acetyl-4 aminophenol) is one of the most widely used analgesics and antipyretics in the world.4 Paracetamol is a non-opioid agent, and it is believed that it primarily acts on the central nervous system by way of central cyclooxygenase inhibition, and it probably has an indirect influence on the serotoninergic system. It has a good safety profile and easily passes through the blood-brain barrier, which assures it has an effective analgesic.5 Opioids are frequently preferred in curettage procedures, and one of the most commonly administered opioids is fentanyl. In general, fentanyl is administered in a dosage of 1 μg/kg during curettage procedures.6 Our study was carried out to determine whether the combined administration of fentanyl (1 μg/kg) and propofol or paracetamol (20 mg/kg) and propofol would display similar effects in providing adequate analgesia, and patient and surgeon satisfaction, resulting in a comparable recovery profile. BODY.METHODS: We performed a prospective, randomized clinical study in January 2015 at Kanuni Sultan Süleyman Education and Training Hospital, Istanbul, Turkey. The study protocol was approved by the Kanuni Sultan Süleyman Education and Training Hospital Ethics Committee in Istanbul, Turkey. Our study is in accordance with the principles of Helsinki Declaration. Patients who were scheduled for suction termination procedures, fulfilled the inclusion criteria and provide their written informed consent were divided into 2 groups using a computer-generated randomizing system. The informed consent forms were obtained from 148 women scheduled for suction curettage in the Istanbul Kanuni Sultan Suleyman Education and Training Hospital between January and February 2015. Patients were randomized into either a fentanyl (1 μg/kg) group or a paracetamol (20 mg/kg) group. Two patients refused anesthesia in the paracetamol group. Thus, 146 patients were enrolled in the study (fentanyl group: 76 [52.1%] and paracetamol group: 70 [47.9%]). The exclusion criteria were: 1) American Society of Anesthesiologists (ASA) scores7 above II, 2) a history of allergic reactions to paracetamol or morphine, 3) a history of paracetamol, opioids or non-steroid anti-inflammatory drug (NSAIDs) use in the 48 hours before requiring chronic analgesic treatment, 4) chronic alcoholism, deficiency of liver and kidney, 5) cardiovascular disease, 6) bleeding diathesis, 7) <18 years of age, 8) body mass index >30 kg/m2, and 9) a history of neurological disease, regular sedative medication, and substance abuse. Patients were taken into the operating room, where an intravenous (IV) cannula was inserted. Monitoring consisted of peripheral pulse oximetry, non-invasive blood pressure measurements at 5-minute intervals, and electrocardiography (ECG). After IV propofol (1 mg/kg) was given to each patient, the patients received either an IV fentanyl bolus (1 μg/kg; fentanyl group) or an IV paracetamol bolus (20 mg/kg; paracetamol group) according to the randomization. We recorded the modified Aldrete score, visual analogue scale (VAS), procedure time, and any adverse events such as bradycardia, hypotension, nausea, and vomiting. The modified Aldrete scoring system (Table 1) was used to measure the patients' recovery time.8 Patients were observed for 60 minutes after the curettage procedure. For postoperative pain assessment, the VAS was used (VAS: 0-10; 0: no pain, 10: worst pain imaginable). Intraoperatively, systolic and diastolic blood pressure, heart rate (HR), and peripheral oxygen saturation (SpO2) values were recorded at 0, 5, 10, 15, and 20 minutes. Table 1 Demographic data of 146 women (fentanyl group: 76 and paracetamol group: 70) who were scheduled for suction curettage. Measurements of blood pressure, HR, and SpO2 were measured at pre-induction of fentanyl and paracetamol, at 5 minutes after induction, and at 5, 10, 15, and 20 minutes following the completion of the procedure. Also, the durations of the surgical procedure and anesthesia were recorded. For statistical analysis of the data, Number Cruncher Statistical System (NCSS) 2007 (Kaysville, Utah, USA) was used. When evaluating the study data, in addition to the descriptive statistical methods (mean, standard deviation, median, frequency and rate), student's t-tests were used for intergroup comparisons of normally distributed variables and the Mann-Whitney U tests were used for intergroup comparisons of data that was not normally distributed. A p-value of <0.05 was accepted as statistically significant. BODY.RESULTS: There were 146 patients involved in this study. When the demographic data, ASA and operation values of the 146 patients were compared (Figure 1), no significant differences were determined between groups (p>0.05) (Table 1). Also, no significant differences were found between the 2 groups in terms of VAS and modified Aldrete scores after 60 minutes (p>0.05) (Table 2). There was no significant difference in the HR between groups (p>0.05) (Figure 2). In the fentanyl group, systolic blood pressures at 5, 10, 15, and 20 minutes were significantly lower (p<0.05) than in the paracetamol group; however, there was no difference between groups at 0 minutes (p>0.05) (Figure 3). Diastolic blood pressures at 5 minutes were significantly lower (p<0.05) in the fentanyl group; however, there were no differences between groups at 10, 15 and 20 minutes (p>0.05) (Figure 4). In the fentanyl group, SpO2 at 5 minutes was significantly lower (p<0.05) than in the paracetamol group, but there were no differences between groups at 10, 15, and 20 minutes (p>0.05) (Figure 5). No significant differences were found between groups with respect to side effects. Figure 1Flow diagram of 146 women who were scheduled for suction curettage. SBP - systolic blood pressure, DBP - diastolic blood pressure, HR - heart rates, VAS - visual analogue scale Table 2 Visual analogue scale (VAS) and modified Aldrete scores of the patients at 60th minutes ( p >0.05). Figure 2Heart rates of 146 women who were scheduled for suction curettage. min- minutes Figure 3Systolic blood pressures of 146 women who were scheduled for suction curettage. min- minutes Figure 4Diastolic blood pressures of 146 women who were scheduled for suction curettage. min- minutes Figure 5Oxygen saturation (SpO2) of 146 women who were scheduled for suction curettage. min- minutes BODY.DISCUSSION: Effective pain management is an important component of ambulatory anesthesia. This study demonstrates that 20 mg/kg paracetamol is a safe and effective intravenous nonopioid analgesic for the treatment of postoperative pain management in patients recovering from dilatation and curetage procedure. In our study, no significant differences were found between the 2 groups in terms of 60th minutes VAS and we did not observe any adverse effects. Gynecological patients have the highest unplanned admission rate in our hospital (85%). Ineffective poorly controlled pain, post-operative nausea and vomiting (PONV), and acute urinary retention are causes resulting in delay in discharge patients after ambulatory gynecologic procedures especially for suction termination procedures. We have shown that the analgesic properties and usefulness of IV paracetamol are similar to those of IV fentanyl in suction termination procedures. In our study, no significant differences were found between the 2 groups in terms of 60-minute VAS results and we did not observe any adverse effects. Fentanyl and remifentanyl narcotics are commonly used for perioperative pain relief in brief surgical procedures as these drugs provide good analgesia. Opioid analgesics are effective, but have troublesome and potentially dangerous side-effects, and their potential for abuse may lead to regulatory and logistical difficulties. Fentanyl depress ventilation, induce chest wall rigidity, which might constitute delayed awakening. This effect can delay recovery period. Fentanyl has fewer side-effects, but can still produce dose-dependent respiratory depression, which may contribute to delayed awakening, bradycardia, and hypotension.9 These effects can delay discharge in day case procedures. Paracetamol is used as a non-opioid analgesic. It is an effective and safe drug for managing mild to moderate pain. The IV form of paracetamol passes easily through the blood-brain barrier and shows its central analgesic effects within 15-20 minutes, which start to decline 4 hours after administration. Thus, paracetamol is preferred in most surgical patients for it does not affect mental status, bleeding, respiratory drive, gastric mucosa integrity, or renal function.10 Ali et al9 compared paracetamol and fentanyl for pain relief in dilatation and curettage procedures. They found an insignificant difference between groups and demonstrated that IV paracetamol may be as effective as fentanyl. However, there are not enough studies on this topic, and further large-scale studies are required. Therefore, we undertook this prospective study to examine IV paracetamol as a suitable alternative to fentanyl for perioperative pain, hemodynamic changes and postoperative complications in suction termination of first-trimester pregnancies. Alhashemi et al11 compared IV paracetamol and oral ibuprofen in patients undergoing lower segment cesarean sections, as the analgesic supplementation agent to morphine. Their results indicated that patients in the IV paracetamol group had better pain control compared to the ibuprofen group. Sinatra et al12 compared IV paracetamol with a placebo and found that IV paracetamol administered over a 24-hours period in patients with moderate to severe pain after orthopedic surgery provided rapid and effective analgesia and was well-tolerated. Kouchek et al10 compared paracetamol and fentanyl in intensive care unit patients and found similar pain scores between the two groups and any adverse effects were observed. In our study, no significant differences were found between the 2 groups at the 60-minute VAS, and we did not observe any adverse effects. Craig et al13 compared IV paracetamol and IV morphine for acute limb trauma in an emergency department. The patients received either 1,000 mg IV paracetamol or 10 mg IV morphine. There was no significant difference in the rescue medication, but there were significantly more adverse reactions in the morphine group. There were also no significant differences between the groups in mean pain scores and patient satisfaction. Khan et al14 examined 84 patients undergoing outpatient knee arthroscopy and compared pain scores and adverse reactions between IV paracetamol (1,000 mg) and IV morphine (0.1 mg/kg) given prior to awakening from general anaesthesia. There was no difference in pain scores between the patients, but there were more adverse reactions, dizziness, nausea, and vomiting in the morphine patients. This study has important implications for discharge times from outpatient surgical hospitals. In many studies, the 2 groups also showed no differences in terms of noninvasive hemodynamic variables.6,9,10 When we compared group HRs, there was no significant difference between the groups. In the fentanyl group, systolic blood pressures at 5, 10, 15 and 20 minutes were significantly lower, and the diastolic blood pressure at 5 minutes was significantly lower. Furthermore, in the fentanyl group, SpO2 at 5 minutes was significantly lower. Küçük et al6 compared the administration of fentanyl (1 μg/kg) and fentanyl (0.5 μg/kg) in dilation and curettage procedures. In the fentanyl 0.5 μg group, modified Aldrete scores at postoperative 5 and 10 minutes were significantly higher. In our study, no significant differences were found postoperatively between the 2 groups in terms of modified Aldrete scores at 60 minutes. BODY.DISCUSSION.STUDY LIMITATIONS: Small sample size, lack of placebo arm, single blinded nature, short follow up duration and targeted to a specific population of ambulatory surgeries were reasons we could not draw further conclusions as to the other ambulatory anesthetic procedures such as hysteroscopy, colposcopy, cryotherapy, cone biopsy, and endometrial biopsy. In conclusion, during moderately painful ambulatory surgical procedures, IV paracetamol may be an asset against opioid analgesics in the control of acute postoperative pain.

TITLE: Pharmacokinetics, Safety, and Tolerability of GLPG0259, a Mitogen-Activated Protein Kinase-Activated Protein Kinase 5 (MAPKAPK5) Inhibitor, Given as Single and Multiple Doses to Healthy Male Subjects ABSTRACT.BACKGROUND AND OBJECTIVES: GLPG0259 is a small-molecule inhibitor of mitogen-activated protein kinase-activated protein kinase 5 (MAPKAPK5), a kinase enzyme that plays a role in important inflammatory pathways. The main objectives of the phase I clinical studies in early development were to characterize the pharmacokinetics, tolerability, and safety of GLPG0259 in healthy subjects, including the development of a solid dosage form (free-base pellets and fumarate salt capsules) and the potential for interaction of GLPG0259 with methotrexate. ABSTRACT.SUBJECTS AND METHODS: Four phase I studies were initiated. Study 1 was a randomized, double-blind, placebo-controlled study to evaluate the safety, tolerability, and pharmacokinetics of single ascending doses (1.5–150 mg) and multiple oral doses (20 and 50 mg once daily) of GLPG0259 in healthy male subjects (n = 34). Study 2 was a randomized, double-blind, placebo-controlled study to evaluate the safety, tolerability, and pharmacokinetics of oral multiple ascending doses of GLPG0259 (25–75 mg once daily) given for 14 days to healthy male subjects, and to get preliminary information on the potential pharmacokinetic interaction between GLPG0259 and methotrexate (n = 24). Studies 3 and 4 were open-label, randomized, crossover studies to compare the oral bioavailability of two solid dosage forms of GLPG0259 (a capsule) relative to an oral solution after a 100 mg or 50 mg single dose and to evaluate the effect of food on these formulations (n = 12 for each study). ABSTRACT.MAIN OUTCOME MEASURES: The non-compartmental pharmacokinetic parameters for plasma concentrations of GLPG0259 were determined, and a population pharmacokinetic model of GLPG0259 was developed to support the planning of the number and timing of the sparse samples to be taken per patient in the phase II study. Safety and tolerability data are also summarized. ABSTRACT.RESULTS: The absorption of GLPG0259 was slow, with a decrease in the absorption rate with increasing dose, and there was decreased elimination, with an apparent terminal elimination half-life of 26.0 hours. On the basis of statistical analysis of variance, the exposure to GLPG0259 increased in proportion to the dose over a 30–150 mg single-dose range and a 25–75 mg repeated-dose range. Between- and within-subject variability in GLPG0259 pharmacokinetics was low/moderate (coefficient of variation [CV] 16–30%). After once-daily repeated dosing, steady-state plasma concentrations were reached at between 5 and 8 dosing days, which is consistent with the long apparent elimination half-life of GLPG0259. Food increased the bioavailability of GLPG0259 given in a solid dosage form. Co-administration of GLPG0259 with a single dose of methotrexate 7.5 mg did not result in any change in the pharmacokinetic profiles of either GLPG0259 or methotrexate. ABSTRACT.CONCLUSION: In summary, the investigation of safety/tolerability and pharmacokinetics in the early development phase showed that single and repeated doses of GLPG0259 were safe and well tolerated. The most common adverse event reported was mild gastrointestinal discomfort. The pharmacokinetics characterized in healthy male subjects showed no major obstacles and supports a once-daily oral regimen in patients. BODY.INTRODUCTION: Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory and joint degenerative disease, which affects almost 1% of the adult population worldwide, with onset classically occurring between the ages of 30 and 50 years, and a higher prevalence in women. The disease is characterized by pain, stiffness, and restricted mobility due to persistent symmetrical inflammation of the synovial membranes of multiple joints, which ultimately results in irreversible damage of the joint cartilage and bone.[1–3] Development of the disease involves an inflammatory response of the synovial membrane, accompanied by infiltration of a variety of immune cells, which leads to the build-up and maintenance of a cytokine network. One of the cytokines central to this network is tumor necrosis factor (TNF), as is clearly demonstrated by the clinical success of TNF blockers in treating RA. TNF and other proinflammatory cytokines contribute to cartilage and bone erosion by inducing release of degradative enzymes, such as matrix metalloproteinases (MMPs), and stimulating the release of receptor-activated NFκB-ligand (RANKL), which triggers differentiation of hematopoeitic cells into bone-resorbing osteoclasts. When left untreated, the disease leads to significant disability associated with high economic costs. In recent years, the therapeutic management of patients with RA has undergone major evolution. Up to 10 years ago, therapeutic approaches relied on synthetic disease-modifying anti-rheumatic drugs (DMARDs) such as methotrexate and sulphasalazine, which had only partial clinical benefit and were associated with significant toxicity. A considerable advance in the effective treatment of RA came from the introduction of the biologic therapeutics that neutralize cytokines or their receptors (TNFα and interleukin [IL]-6) or that inhibit cellular activation (B-cell or T-cell activation).[4,5] However, because of the high production costs, inconvenience of parenteral administration, increased risk of infections, and potential immunogenicity of biologics, there is still a need for less expensive and orally administered drugs.[4] Hence, the development of small-molecule inhibitors targeting disease-relevant signal transduction pathways is being pursued by various companies.[6] GLPG0259 is a small-molecule inhibitor of mitogen-activated protein kinase–activated protein kinase 5 (MAPKAPK5), a kinase enzyme that plays a role in important inflammatory pathways. Inhibition of MAPKAPK5 with GLPG0259 represents a novel mechanism of action in the treatment of RA. MAPKAPK5 belongs to a family of mitogen-activated protein kinases that play an important role in several cellular processes, including inflammation, proliferation, and differentiation. MAPKAPK5 is involved in a transduction pathway in RA patients that ultimately leads to secretion of catabolic enzymes such as MMP1, which cause damage to the bone and cartilage in these patients. GLPG0259 is a potent inhibitor of MAPKAPK5, and in vitro it reduces the release of several mediators of inflammation and bone degradation, such as MMP1, MMP13, TNF, and IL6. After oral administration in mice, GLPG0259 reduces paw inflammation as well as bone destruction in the mouse collagen-induced arthritis (CIA) model of human RA at a dose of 1 mg/kg and higher. Even in mice with late-stage CIA disease, GLPG0259 reduces inflammation and bone destruction. The main objectives of the phase I clinical studies in early development were to characterize the pharmacokinetics, tolerability, and safety of GLPG0259 in healthy subjects, including the development of a solid dosage form and the potential for interaction of GLPG0259 with methotrexate (the anchor drug in RA patients). However, an exploratory phase II study in a small number of RA patients with an insufficient response to methotrexate showed no significant clinical benefit of GLPG0259 compared with placebo, and the development of GLPG0259 was discontinued (Westhovens R et al., unpublished data).[7] BODY.SUBJECTS AND METHODS: All studies were conducted in accordance with accepted standards for the protection of subject safety and welfare, and the principles of the Declaration of Helsinki and its amendments, and were in compliance with Good Clinical Practice. Protocols and informed consents were approved by the Ziekenhuis Netwerk Antwerpen (ZNA) Institutional Review Board (Antwerp, Belgium). All healthy participants gave written informed consent prior to study initiation. BODY.SUBJECTS AND METHODS.STUDY DESIGNS.STUDY 1: FIRST-IN-HUMAN, SINGLE ASCENDING AND MULTIPLE ORAL DOSES: This was a randomized, double-blind, placebo-controlled, single-center study to evaluate the safety, tolerability, and pharmacokinetics of single ascending and multiple oral doses of GLPG0259 in healthy subjects. Eligible subjects (aged 18–50 years, body mass index [BMI] 18–30 kg/m2) were in good health with no clinically significant deviation from normal in terms of medical history, physical examinations, electrocardiograms (ECGs), or clinical laboratory determinations. Subjects were excluded from the study if they had medical history of abnormal platelet function or a history of a current immunosuppressive condition. The study was divided into two parts: Part 1 (n = 16 subjects): Single escalating dose intake of GLPG0259 or matching placebo (1.5–150 mg) was studied in two cohorts of eight subjects (active or placebo in a 3 : 1 ratio) in an alternating panel design. There was a minimum 7-day washout period between treatments for each subject within a cohort and at least 3 days for each dose increment between cohorts.Part 2 (n = 18 subjects): Multiple dose intakes (20 mg and 50 mg once daily) of GLPG0259 or a matching placebo over 5 days were studied in two cohorts of nine subjects (active or placebo in a 2 : 1 ratio). There was a minimum period of 7 days between the two cohorts. Treatment allocation was determined by a computer-generated randomization schedule. Subjects were admitted to the clinical unit on the evening prior to dosing (day -1) and were confined until 24 hours after the last dose. In both parts, GLPG0259 free-base solution (1–10 mg/mL in 40% [w/v] hydroxypropyl-ß–cyclodextrin, pH 3) or a matching placebo was given using a graduated syringe. A volume of 200 mL of water was given to each subject immediately at the time of dosing. Treatments were administered after a standard breakfast (i.e. four slices of whole-wheat bread, one slice of salami, one slice of cheddar cheese, one tablespoon of butter and jam, totaling 590 kilocalories) except during the last dosing period of part 1, where the treatment was administered after an overnight fast to assess the food effect on GLPG0259 bioavailability. Drinks were standardized to at least 1000 mL of mineral water per day. Blood samples for pharmacokinetics were collected at regular intervals over 24 hours (part 1: 1.5–15 mg; part 2: 20 mg and 50 mg on day 1), 4 days (part 1: 30–150 mg), or 7 days postdose (part 2: 20 mg and 50 mg on day 5). Blood was collected in tubes containing lithium heparinate as an anticoagulant in order to obtain plasma for analysis of the concentrations of GLPG0259. Within 30 minutes after blood collection, the plasma was separated in a refrigerated centrifuge (4–8°C) for 10 minutes at approximately 1500 g, transferred into two polypropylene tubes with at least 500 μL of plasma per tube, and stored at -20°C until analysis. Three urine fractions were collected in part 2 on days 1 and 5 over a 24-hour period to determine the amount of GLPG0259 excreted in urine. After homogenization and recording of the total weight, two 10 mL samples for the GLPG0259 assay were stored at or below -20°C until analysis. BODY.SUBJECTS AND METHODS.STUDY DESIGNS.STUDY 2: MULTIPLE ASCENDING ORAL DOSES AND METHOTREXATE DRUG-DRUG INTERACTION: This was a phase I, randomized, double-blind, placebo-controlled, single-center study to evaluate the safety, tolerability, and pharmacokinetics of oral multiple ascending doses of GLPG0259 given for 14 days to healthy subjects (n = 24), and to get preliminary information on the potential pharmacokinetic interaction between GLPG0259 and methotrexate. The criteria for subject eligibility were the same as those listed for study 1. A total of 24 healthy male subjects were randomized into three cohorts of eight subjects dosed orally once daily with either placebo or GLPG0259 25 mg, 50 mg, or 75 mg (active or placebo in a 3 : 1 ratio). In addition, the subjects from the 50 mg cohort received one single dose of methotrexate 7.5 mg on two occasions: 7 days prior to dosing with GLPG0259 (on day -7) and on day 14 at the same time as the last GLPG0259 dose. GLPG0259 free base (10 mg/mL in 40% [w/v] hydroxypropyl-ß–cyclodextrin, pH 3) or a matching placebo was administered once daily for 14 days, using a syringe, as for study 1. Subjects in the 50 mg dose group were additionally administered an oral dose of methotrexate 7.5 mg (3 tablets of Ledertrexate® 2.5 mg; Wyeth-Pfizer) on two occasions. A dose of 4 mg of folic acid (Folavit®; Kela Pharma NV) was administered 24 hours after each methotrexate administration as a preventive measure for methotrexate toxicity. Folic acid was administered after all safety and pharmacokinetic assessments had been done. Blood samples for pharmacokinetics were collected at regular intervals over 24 hours (on days 1 and 13 [in the 50 mg cohort only]) or over 7 days after the last dose on day 14 (i.e. up to day 21) to assess plasma concentrations of GLPG0259. Blood sample handling was similar to that described for study 1. For methotrexate (in the 50 mg cohort only), blood samples were collected at regular intervals over 24 hours (on day -7 and day 14) in tubes containing lithium heparinate, in order to obtain plasma, and were stored at -20°C until analysis. BODY.SUBJECTS AND METHODS.STUDY DESIGNS.STUDY 3: ORAL RELATIVE BIOAVAILABILITY AND THE FOOD EFFECT: This was a phase I, open-label, randomized, three-period, three-treatment crossover study to compare the oral bioavailability of a solid dosage form of GLPG0259 (a capsule) relative to an oral solution, and to evaluate the effect of food on oral bioavailability of GLPG0259 formulated as a capsule in healthy subjects (n = 12). The criteria for subject eligibility were the same as those listed for study 1. The treatments consisted of an oral dose of a 50 mg GLPG0259 free-base solution given after an overnight fast (treatment A), a GLPG0259 fumarate capsule (equivalent to 50 mg free base) given after an overnight fast (treatment B), and a GLPG0259 fumarate capsule (equivalent to 50 mg free base) given 30 minutes after the start of a high-fat, high-calorie breakfast (treatment C). Each subject was administered treatments A, B, and C in one of the two treatment sequences (i.e. ABC or ACB) determined by a computer-generated randomization schedule. There was at least a 7-day washout period between treatments for each subject. Subjects were admitted to the clinical unit on the evening prior to dosing (day -1) and were confined until 24 hours after the last dose. For treatment A, GLPG0259 free base was administered as 5 mL of 10 mg/mL in 40% (w/v) hydroxypropyl-ß–cyclodextrin (pH 3), using a syringe. A volume of 235 mL of water was given to each subject immediately at the time of dosing. Capsules to be administered for treatments B and C were filled with 50 mg of GLPG0259 as a fumarate salt. One capsule was given with 240 mL of water alone (treatment B) or after a high-fat, high-calorie breakfast as described in the FDA Guidance for Industry[8] (i.e. two eggs fried in butter, two slices of bacon, two slices of toast with butter, 113 g of hash brown potatoes, and 240 mL of whole milk, totaling 800–1000 kilocalories). The subjects took the 50 mg capsule with 240 mL of water, within 10 minutes after the high-fat, high-calorie breakfast. The breakfast had to start 30 minutes prior to administration of the study drug, and the subjects had to eat their breakfast within 20 minutes. Blood samples for pharmacokinetics were collected at regular intervals over 96 hours to assess plasma concentrations of GLPG0259. Blood sample handling was similar to that described for study 1. BODY.SUBJECTS AND METHODS.STUDY DESIGNS.STUDY 4: ORAL RELATIVE BIOAVAILABILITY OF TWO SOLID DOSAGE FORMS: This was a phase I, randomized, open label, two-period, two-treatment crossover study to compare the oral bioavailability of two solid oral formulations of GLPG0259 after single-dose intake in healthy subjects (n = 12). The criteria for subject eligibility were the same as those listed for study 1. The two treatments consisted of an oral dose of two fumarate capsules containing GLPG0259 (equivalent to 25 mg free base) given exactly 30 minutes after the start of a high-fat, high-calorie breakfast (treatment A) and a single free-base pellet capsule containing GLPG0259 50 mg given exactly 30 minutes after the start of a high-fat, high-calorie breakfast (treatment B). Each subject was administered treatments A and B in one of the two treatment sequences (i.e. AB or BA) determined by a computer-generated randomization schedule, with at least a 10-day washout period between treatments. Subjects were admitted to the clinical unit on the evening prior to dosing (day -1) and were confined until 24 hours after the last dose. Capsules administered in fed conditions were taken within 10 minutes after the high-fat, high-calorie breakfast, as in study 3. Blood samples for pharmacokinetics were collected at regular intervals over 96 hours to assess plasma concentrations of GLPG0259. Blood sample handling was similar to that described for study 1. BODY.SUBJECTS AND METHODS.SAFETY ASSESSMENTS: In all four studies, general safety was evaluated by the incidence of adverse events (AEs) through non-leading questioning, clinical laboratory parameters (hematology, biochemistry, and urinalysis), vital signs, 12-lead ECGs, and physical examinations. BODY.SUBJECTS AND METHODS.BIOANALYTIC AND PHARMACOKINETIC METHODS.GLPG0259: Plasma GLPG0259 concentrations were determined using a validated liquid-chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) assay. In brief, the internal standard (deuterated GLPG0259; 20 μL at 0.25 μg/mL) was added to plasma samples and then processed by liquid–liquid extraction. The evaporated and reconstituted samples were injected into a Sciex API 4000TM LC–MS/MS equipped with a short high-pressure liquid chromatography (HPLC) column. GLPG0259 was detected with multiple reaction monitoring. Quantification was performed using peak area ratios and standard curves (with 1/x2 linear least-squares regression) prepared from calibration standards. The lower limit of quantification was 0.200 ng/mL. The between- and within-run precision for quality controls, expressed as coefficients of variation (CVs), were no greater than 13.9% and 7.50%, respectively, with deviations from nominal concentrations of no more than 12.0%. A method adapted from the plasma bioanalytic method was used to determine the concentrations of GLPG0259 in urine. The internal standard (deuterated GLPG0259; 20 μL at 0.5 μg/mL) was added to 20 μL of the urine sample. The corresponding solution was diluted 50-fold and injected directly into a Sciex API 4000TM LC–MS/MS. The lower limit of quantification was 2.00 ng/mL. The within-run precision for quality controls, expressed as the CV, was no greater than 6.7%, with deviations from nominal concentrations of no more than 6.5%. Plasma GLPG0259 concentrations were analyzed by a non-compartmental method. The maximum plasma drug concentration (Cmax) and time to reach Cmax (tmax) values were observed directly from the data. The terminal elimination rate constant (λz) was determined by log-linear regression analysis of the elimination phase. The apparent terminal elimination half-life (t1/2,λz), calculated as t1/2,λz = Ln2/λz, was reported only if more than three datapoints were used for linear regression to determine λz with an adjusted r2 value of ≥0.900. Area under the plasma concentration–time curve (AUC) values over the collection interval (AUCt), over the dosing interval (AUCτ), or extrapolated to infinity (AUC∞) were determined using standard non-compartmental methods (WinNonLin® version 5.2 software; Pharsight Corporation, Mountain View, CA, USA). The relative bioavailability (Frel) was calculated as the ratio between the AUCs for the test formulations (fumarate capsules or free-base pellet capsules) and the AUCs for the reference formulations (solution or fumarate capsules) from studies 3 and 4. After multiple dosing, the accumulation of GLPG0259 was estimated as the ratio between the steady-state AUCτ and the day 1 AUCτ (Rac(AUC)). The following urine parameters were determined after multiple dosing for 5 days (study 1 part 2): the amount of GLPG0259 excreted unchanged in urine (Ae24h), expressed as a percentage of the dose, and renal clearance (CLR) over 24 hours (CLR24h). BODY.SUBJECTS AND METHODS.BIOANALYTIC AND PHARMACOKINETIC METHODS.METHOTREXATE: Plasma methotrexate concentrations were determined using a validated LC–MS/MS assay. In brief, the internal standard (deuterated GLPG0259; 200 μL at 25 ng/mL) was added to plasma samples and then processed by liquid–liquid extraction. The evaporated and reconstituted samples were injected into a Sciex API 4000TM LC–MS/MS equipped with a short HPLC column. Methotrexate was detected with multiple reaction monitoring. Quantification was performed using peak area ratios and standard curves (with 1/x2 linear least-squares regression) prepared from calibration standards. The lower limit of quantification was 5.00 ng/mL. The between- and within-run precision for quality controls, expressed as CVs, were no greater than 7.40% and 8.16%, respectively, with deviations from nominal concentrations of no more than 8.0%. The plasma methotrexate concentrations were analyzed by a non-compartmental method, and the following parameters were assessed: Cmax, tmax, t1/2,λz, AUCt, and AUC∞. BODY.SUBJECTS AND METHODS.STATISTICAL ANALYSES: All statistical analyses were conducted using SAS® version 9.1 software (SAS Institute Inc., Cary, NC, USA). For the pharmacokinetic analyses of the four clinical studies, the descriptive statistics analysis included arithmetic means and CVs for Cmax, AUC, t1/2,λz, Ae24h, and CLR24h; the medians and ranges for tmax; and the geometric means and CVs for Rac(AUC) and Frel. Clinical safety was addressed by assessing AEs, physical examinations, laboratory assessments, ECGs, and vital sign results in a descriptive manner. Descriptive statistics and shift tables (according to normal ranges) were calculated for each parameter at every timepoint and in each treatment group. A treatment-emergent AE analysis was performed. The following inferential statistics were performed for each study, with a statistical significance level of p < 0.0500. BODY.SUBJECTS AND METHODS.STATISTICAL ANALYSES.STUDY 1: Dose proportionality was tested on dose-normalized and natural log–transformed GLPG0259 parameters (Cmax normalized to a 1 mg dose [Cmax/dose] and AUC from 0 to 24 hours [AUC24h] normalized to a 1 mg dose [AUC24h/dose]) after single fed dosing by means of mixed-effects analysis of variance (ANOVA) with the cohort and dose as fixed effects and the subject (nested within the cohort) as a random effect. In the case of a significant dose effect being observed on the parameters listed above, comparison between doses was performed using Tukey's test. The tmax, being a discrete variable, was analyzed using a non–parametric Kruskal-Wallis test to assess the dose proportionality. For part 2, a mixed-effects ANOVA was performed on natural log–transformed GLPG0259 parameters (Cmax/dose, AUC24h/dose, t1/2,λz, Ae24h, and CLR24h) with the day, dose, and day-by-dose interaction as fixed effects and the subject as a random effect. Dose proportionality for Rac(AUC) was evaluated from the adapted mixed-effects ANOVA of AUC24h/dose. A Wilcoxon–Mann-Whitney non-parametric test was used to assess the dose proportionality of tmax. The time to reach steady state was assessed by visual inspection of the trough plasma drug concentrations as well as by means of a mixed-effects ANOVA on Ln-transformed GLPG0259 trough plasma drug concentrations. Comparison between days was performed using Tukey's test. The food effect was assessed using geometric mean ratios of the observed pharmacokinetic parameters (Cmax, AUC24h, AUC∞, and t1/2,λz) for GLPG0259, with and without food, and the corresponding 90% confidence intervals (CIs) for the ratios. Calculation of 90% CIs for the geometric mean ratios was based on a mixed-effects model for the natural log–transformed parameters, with the dietary condition as a fixed effect and the subject as a random effect. A Wilcoxon–Mann-Whitney non-parametric test was used to assess the food effect on tmax. BODY.SUBJECTS AND METHODS.STATISTICAL ANALYSES.STUDY 2: Dose proportionality of GLPG0259 pharmacokinetics and steady-state assessment were tested using the same statistical model as described for study 1 part 2. The effect of GLPG0259 on methotrexate pharmacokinetics (day 14 versus day -7) and the effect of methotrexate on GLPG0259 pharmacokinetics (day 14 versus day 13) were separately assessed on natural log–transformed parameters (Cmax, tmax, AUC, and t1/2,λz), using a mixed-effects ANOVA model with the day as a fixed effect and the subject as a random effect. The geometric mean ratio (i.e. the point estimate) of these pharmacokinetic parameters between days 14 and 13 for GLPG0259 and between days 14 and -7 for methotrexate was estimated from this model, using the least-squares mean (LSM) together with the 90% CI. BODY.SUBJECTS AND METHODS.STATISTICAL ANALYSES.STUDIES 3 AND 4: For both studies, the comparison between treatments was assessed on Ln-transformed parameters (Cmax, AUC24h, AUC∞, and t1/2,λz) by means of a mixed-effects ANOVA. The point estimate was calculated as the geometric mean of the individual ratios of each parameter for the test/reference treatments and expressed as a percentage. The 90% CI of the point estimates was calculated using the mean square error of the ANOVA. As tmax is a discrete variable dependent on selected blood sampling times, the same comparisons were assessed using a non-parametric test. The 90% non-parametric CIs for the treatment differences were calculated. BODY.SUBJECTS AND METHODS.POPULATION PHARMACOKINETIC MODEL: A population pharmacokinetic model was developed with data from the three first phase I studies (at the time of performing the population pharmacokinetic analysis, study 4 had not been performed yet), which included 54 subjects who received the active treatment within the dose range of 1.5–150 mg on at least one occasion (n = 6 at 1.5, 5, and 15 mg; n = 18 at 20–30 mg; n = 24 at 50 mg; n = 12 at 60–75 mg and 100 mg; n = 6 at 150 mg) as fumarate salt capsules or free-base solution given in either the fasted or fed state. The model that was developed was then used to support the planning of the number and timing of the sparse samples to be taken per patient in the 3-month phase II study. An exploratory graphical analysis of the pharmacokinetics of GLPG0259 was performed. The graphical analysis consisted of plotting and comparing individual profiles and the smoothes of dose-normalized profiles. Dose linearity was evaluated by comparing the dose-normalized profiles. The exploratory graphical analysis plots were also scrutinized for food and formulation effects. All analyses were performed in accordance with appropriate guidelines.[9,10] The population pharmacokinetic analyses were performed using NONMEM® version 7.1.0 software.[11] The NONMEM® model fitting used the first-order conditional estimation (FOCE) method with the interaction option throughout. S-Plus version 6.2 software was used for exploratory graphical analysis. R software (version 2.12.2)[12] was used for evaluation of goodness of fit and model evaluation. The program WinPOPT (version 1.2.1)[13] was used to aid selection of the timing and number of samples to be taken per patient in phase II. BODY.RESULTS.SAFETY AND TOLERABILITY: With the exception of a single subject who discontinued study 1 because of a nephrolithiasis while on placebo (reported as a serious AE [SAE]), all subjects completed the studies. The four studies showed a consistent pattern of AEs. Nausea, abdominal discomfort, and loose stools were the most frequently reported AEs, showing a dose-related pattern of incidence and severity from a dose of 50 mg upward. The feeding status or type of formulation had no influence on these AEs. All other AEs were typical phase I environment events, such as somnolence, fatigue, headache, oropharyngeal pain, and nasopharyngitis. No clinically relevant trends or changes were observed in the median laboratory and urinalysis values over time. A single case of a mild alanine aminotransferase increase was observed in a subject at the 75 mg dose in the second study. Across the four studies, no clinically relevant trends or changes were observed in the median vital sign values and ECG parameters over time. No treatment-emergent abnormalities related to vital signs or ECG parameters were observed in more than one subject during the trials. None of the abnormalities related to vital signs or ECG parameters were considered clinically relevant by the investigators. After multiple dosing, the maximum tolerated dose was established as being 50 mg once daily. BODY.RESULTS.SAFETY AND TOLERABILITY.GLPG0259 SINGLE-DOSE PHARMACOKINETICS (STUDY 1): GLPG0259 plasma concentration–time data are plotted in figures 1 and 2 (linear and semi-logarithmic plots), and the pharmacokinetic parameters are listed in table I. At the three lowest doses (up to 15 mg), λz could not be reliably estimated in most of the subjects, because of insufficient datapoints to characterize the terminal elimination phase. In addition, for some subjects at the highest doses (≥30 mg), the AUC∞ was poorly estimated, with an extrapolated AUC from 24 hours to infinity that represented more than 20% of the total AUC. Consequently, the t1/2,λz and AUC∞ of these subjects were not included in the summary statistics, and no inferential statistical analysis was performed on these two parameters. After a single oral administration to healthy, fed subjects, GLPG0259 was absorbed slowly, with the median tmax increasing with the dose from 2 to 7 hours (table I). The terminal plasma elimination phase of GLPG0259 was parallel for doses ≥30 mg and displayed a monophasic profile (figure 1). Table IGLPG0259 pharmacokinetic parameters after a single oral dose in fed healthy subjects (n = 6 per dose group) Fig. 1Mean (± standard error of the mean) plasma GLPG0259 concentrations after single oral doses in fed healthy subjects (n = 6 per dose group): (a) linear and (b) semi-logarithmic plots. Fig. 2Mean (± standard error of the mean) plasma GLPG0259 concentrations after once-daily repeated oral dosing in fed healthy subjects: (a) dosing for 5 days (n = 6 per dose group); (b) dosing for 14 days (n = 6 per dose group). After single dosing, Cmax and AUC24h increased proportionally within the 15–100 mg and 30–150 mg dose ranges (table I). A significant dose effect on tmax was observed, with a higher median value observed at the two highest doses. Although no statistical analysis was performed on t1/2,λz, no noticeable difference in this parameter was observed, with a mean value of about 26.0 hours (range 25.5–26.4 hours). BODY.RESULTS.SAFETY AND TOLERABILITY.GLPG0259 REPEATED-DOSE PHARMACOKINETICS (STUDIES 1 AND 2): GLPG0259 plasma concentration–time data are plotted in figure 2, and the pharmacokinetic parameters are listed in table II. As was already evident from the single-dose pharmacokinetics, GLPG0259 was absorbed slowly, with a trend toward an increase in tmax with increased dosing (table II). Table IIGLPG0259 pharmacokinetic parameters after once-daily repeated oral dosing in fed healthy subjects (n = 6 per dose group) The steady-state GLPG0259 plasma concentration was reached at between 4 and 8 dosing days (figure 2, table III). After the last dose, plasma elimination of GLPG0259 over time displayed a monophasic profile, with a t1/2,λz of about 39 hours (range 35.0–41.6 hours). An approximate 2.5-fold increase in AUC24h and Cmax of GLPG0259, similar for all doses, was observed after once-daily dosing, which was consistent with the long GLPG0259 t1/2,λz. After repeated administration, GLPG0259 did not deviate from dose proportionality, with AUC24h and Cmax increasing in proportion to the dose within the 20–75 mg dose range. Overall, the between-subject variability in AUC24h and Cmax at steady state was low/moderate (between-subject CV range 16–30%) as was the within-subject variability, which was derived from the square root of the mean square error of the ANOVA (the CVs of AUC and Cmax ranged between 9.8% and 20%; data not shown). Table IIITrough plasma GLPG0259 concentrations after once-daily repeated oral dosing in fed healthy subjects (n = 6 per dose group) Excretion of unchanged GLPG0259 in urine was rapid, with about 64–88% excreted within the first 12 hours (data not shown). The Ae24h of GLPG0259 represented 4.99% and 10.4% of the dose administered after single and multiple dosing, respectively, of 50 mg of GLPG0259 for 5 days (table II). The increase in the amount of GLPG0259 excreted in urine between the first and last doses mirrored the accumulation of GLPG0259 observed in plasma. As a consequence, the CLR24h remained constant between the first and last doses. At the 20 mg dose, the increase in Ae24h between the first and last doses (from 3.47% to 4.75%) was smaller than the increase in exposure (from 116 to 261 ng · h/mL), resulting in a slight decrease in CLR with time (from 5.95 to 3.74 L/h). However, these differences were not statistically different and could have been due to high variability in individual Ae24h values (range 0.685–12.0%; CV 81.4%) compared with AUC24h on day 5 (range 197–351 ng · h/mL; CV 19.3%). BODY.RESULTS.SAFETY AND TOLERABILITY.DRUG-DRUG INTERACTION WITH METHOTREXATE (STUDY 2): GLPG0259 and methotrexate plasma concentration–time data are plotted in figure 3, and GLPG0259 and methotrexate pharmacokinetic parameters with summary statistical analyses are presented in table IV. Regarding GLPG0259, co-administration of methotrexate 7.5 mg did not significantly alter the rate and extent of absorption of GLPG0259, with point estimates for Cmax and AUC24h of 102.67% and 102.11%, respectively. Although the t1/2,λz of GLPG0259 could be estimated on one occasion only, there was no modification of the elimination, as shown by the superimposable elimination phases with or without methotrexate in figure 3. It must be noted that even if the study was not powered to analyze the influence of methotrexate on GLPG0259 pharmacokinetics, using the 90% CI approach, the intervals were narrow and their boundaries fell within the 80–125% bioequivalence range for both Cmax and AUC24h (table IV). These results are explained by the low/moderate within-subject variability in GLPG0259 pharmacokinetics (<20%) and suggest that a sample size of 12 subjects would be sufficient to show bioequivalence between two treatments. Table IVSummary statistics for GLPG0259 and methotrexate pharmacokinetic parameters (n = 6) Fig. 3Mean (± standard error of the mean) plasma concentrations of (a) GLPG0259 and (b) methotrexate after administration of each drug alone or in combination to fed healthy subjects (n = 6). The plasma pharmacokinetic parameters of methotrexate observed in this study were in agreement with those reported previously for the methotrexate 7.5 mg dose.[14,15] When methotrexate was co-administered with GLPG0259 50 mg, the rate of absorption of methotrexate was slightly but not statistically significantly decreased, with a point estimate for Cmax of 89.63% (figure 3, table IV). The extent of absorption (AUC∞) and the elimination (t1/2,λz) of methotrexate were not affected by GLPG0259, and their point estimates were 118.22% and 110.64%, respectively. BODY.RESULTS.SAFETY AND TOLERABILITY.BIOAVAILABILITY AND FOOD INTERACTION STUDIES (STUDIES 1, 3, AND 4): As shown in figure 4a, food did not have an impact on the rate and extent of absorption of GLPG0259 given as 100 mg of free-base oral solution, with a Cmax of 31.8 ng/mL (versus 31.0 ng/mL in the fasted state) and an AUC24h of 562 ng · h/mL (versus 572 ng · h/mL in the fasted state) [table V], and corresponding point estimates of 89.67% (90% CI 74.71, 107.61) and 100.42% (90% CI 83.46, 120.83), respectively (table VI). Table VGLPG0259 pharmacokinetic parameters after a single oral dose of GLPG0259 given as various oral formulations to fasted or fed healthy subjects (n = 6 or 12 per formulation) Table VITable VI. Statistical analysis of the food effect on GLPG0259 pharmacokinetic parameters Fig. 4GLPG0259 pharmacokinetic profiles after a single oral dose of GLPG0259 given to healthy subjects as (a) free-base oral solution in the fasted and fed states; (b) free-base oral solution in the fasted state and fumarate salt capsules in the fasted and fed states; or (c) fumarate salt capsules in the fed state and free-base solid dispersion capsules in the fed state. Compared with GLPG0259 free-base oral solution, the bioavailability (both Cmax and AUC∞) of a single 50 mg dose of GLPG0259 given as the fumarate salt in capsule form in the fasted state was decreased by about 45% (table VII). No change in the absorption rate (tmax 6 versus 7 hours) or t1/2,λz (31.6 versus 29.6 hours) was noted. This decrease in bioavailability was prevented by dosing the GLPG0259 fumarate salt capsule in the fed state (figure 4b). In such conditions, the solid dosage form led to bioavailability comparable to that obtained with the GLPG0259 free-base oral solution administered in fasted conditions, as shown by a Cmax of 15.2 ng/mL (versus 12.8 ng/mL) and an AUC∞ of 542 ng · h/mL (versus 536 ng · h/mL) [table V]. Table VIITable VII. Statistical analysis of the formulation effect on GLPG0259 pharmacokinetic parameters Finally, a-head-to-head comparison of two solid dosage forms was investigated after a single 50 mg dose was given in the fed state as capsules of GLPG0259 fumarate salt and GLPG0259 free-base solid dispersion as coated pellets. The two formulations compared well, as shown by Cmax values of 20.4 ng/mL versus 19.8 ng/mL and AUC∞ values of 713 ng · h/mL versus 670 ng · h/mL for the free-base solid dispersion and fumarate salt, respectively (table V), with corresponding point estimates of 103.73% (90% CI 93.73, 114.81) and 107.80% (90% CI 99.76, 116.50), respectively (table VI). Even if these three studies were not powered to compare formulations, using the 90% CI approach, the interval boundaries for both Cmax and AUC were close to or even fell within (study 4) the 80–125% bioequivalence range. BODY.RESULTS.SAFETY AND TOLERABILITY.POPULATION PHARMACOKINETICS OF GLPG0259: The exploratory graphical analysis from study 1 (a single ascending dose) revealed that the elimination of GLPG0259 was independent of the dose, but that the dose-normalized profiles were not superimposable within the entire dose range (1.5–150 mg), that tmax occurred later at higher doses, and that there appeared to be no influence of food on the absorption of the solution formulation. After multiple doses, steady-state GLPG0259 plasma concentrations were reached after 4–5 days. The dose non-linearity observed after single dose administration was not apparent after multiple doses where a smaller dose range of 25–75 mg was given (data not shown). The graphical analysis also showed that the Frel for GLPG0259 was lower for the capsule formulation than for the solution formulation, but that food increased the bioavailability of the capsule to the same level as that of the solution formulation. These findings are in good agreement with the conclusions drawn from traditional pharmacokinetic analysis of the data from these three studies. BODY.RESULTS.STRUCTURAL MODEL DEVELOPMENT: Initially, one- and two-compartment disposition models with simple first-order absorption were compared. Inter-individual variability (IIV) was included on all parameters, and a proportional residual error model was used. The two-compartment model was superior. Next, food and formulation effects were included on Frel, and the absorption sub-model was expanded to a sequential zero- then first-order absorption process for each of the solution and capsule formulations. Ultimately, only the duration of the zero-order input process (D1) differed between the two formulations, followed by the same first-order absorption-rate constant (ka). The statistical model was refined in the next step; IIV was included initially on all structural model parameters but was not able to be estimated on inter-compartmental clearance (Q) and so was removed. Next, inter-occasion variability (IOV) was introduced on Frel, ka, and D1. The introduction of IOV on Frel and D1 resulted in the IIV values for these two parameters being extremely small and considered negligible, and so IIV was removed. The proportional residual error model used at the start of the analysis was found to be adequate and was retained throughout model development. BODY.RESULTS.POPULATION PHARMACOKINETIC MODEL OF GLPG0259: The final population pharmacokinetic model was a two-compartment disposition model with sequential zero- then first-order disposition. The exploratory analysis had clearly shown that dose was a potentially important covariate. The final model contained an influence of dose on the parameters ka and Frel. Dose was included as a covariate on Frel as a power model; Frel increased with increasing dose (figure 5b). Dose was also included as a covariate on ka as a maximum effect (Emax) model; ka decreased with increasing dose up to 50 mg and was then reasonably constant (figure 5a). During the evaluation of dose as a covariate, the parameterization used to describe ka was altered to be equal to λz plus a constant (flip-flop pharmacokinetics). As a result, t1/2,λz could be calculated correctly. Fig. 5Influence of dose on (a) the first-order absorption rate constant and (b) relative bioavailability in the final population pharmacokinetic model. Frel = relative bioavailability; ka = first-order absorption rate constant. Formulation had an effect on D1, which was estimated to be 0.317 hours for the solution formulation and 2.66 hours for the capsule formulation. Formulation also had an effect on Frel, which was estimated to be 0.489 of the Frel for the capsule formulation. The presence of food (a high-fat breakfast) was also found to influence Frel; Frel was 1.89 times greater in the presence of food with the capsule formulation only. Consequently, food was included as a covariate on Frel for the capsule formulation only, and formulation was included as a covariate on Frel and D1. IIV terms were included on the apparent total body clearance (CL/F), apparent volumes of distribution in the central and peripheral compartments (V1/F and V2/F, respectively), and ka. IOV was included on Frel, D1, and ka. A proportional error model was used to describe the residual variability. The parameter estimates for the final population pharmacokinetic model are presented in table VIII. Table VIIIGLPG0259 parameter estimates for the final population pharmacokinetic model The residual variability for the final model (15.0%) was low and showed that the final population pharmacokinetic model described the vast majority of the variability in the data. The value of CL/F estimated for GLPG0259 was 79.3 L/h and was estimated with high precision (relative standard error [SE] 4.0%). The estimate of V1/F was 3030 L and was also precise (relative SE 4.4%). The values for CL/F and V1/F could be used to obtain the t1/2,λz for GLPG0259, which was calculated to be 26.5 hours. In general, all of the parameters associated with the disposition of GLPG0259 were estimated precisely (IIV around 20%). Parameters associated with absorption were less precisely estimated (IIV and IOV ranged between 20% and 75%), indicating that the majority of the overall variability in the pharmacokinetics of GLPG0259 was due to absorption. The value of ka at a dose of 50 mg was calculated to be 0.88/hour. The goodness-of-fit plots for the final population pharmacokinetic model of GLPG0259 are shown in figures 6 and 7. Fig. 6Goodness-of-fit plots: observed data are plotted on the y-axes, and population predictions [graphs (a) and (b)] and individual model predictions [graphs (c) and (d)] are plotted on the x-axes. Graphs () and (c) are on a linear scale, and graphs (b) and (d) are on a logarithmic scale. The dashed datalines are identity lines, and the thick solid datalines are smoothes through the data. The smooth lines lie very close to the identity lines, for both the population and individual predictions, indicating that the structural model describes the data well. IPRED = individual predictions; PRED = population predictions. Fig. 7Goodness-of-fit plots: (a) conditional weighted residuals versus population predictions; (b) absolute individual weighted residuals versus individual predictions; (c) conditional weighted residuals versus time after dose; (d) conditional weighted residuals versus continuous time. The dashed datalines are zero lines, and the thick solid datalines are smoothes through the data. The lack of trends in graphs (), (c), and (d) again indicates that the structural model describes the data well. The lack of a trend in the smooth line in graph (b) shows that the proportional error model is appropriate for describing the residual error. CWRESI = conditional weighted residuals; IPRED = individual predictions; ∣IWRES∣ = absolute individual weighted residuals; PRED = population predictions. Model qualification of the final model, using a visual predictive check (VPC) and a numerical predictive check (NPC), showed that the model was a good description of the data (figure 8). Fig. 8(a) Visual predictive check; (b) numerical predictive check (upper prediction interval limit); and (c) numerical predictive check (lower prediction interval limit). In graph (), the thick solid dataline shows the median of the observed data, and the dark gray shading shows the model-predicted 95% confidence interval around the median. The dotted datalines are the limits between which 95% of the observed data are found, and the light gray shading shows the model-predicted 95% confidence intervals around those limits. In graphs (b) and (c), the thin solid datalines and white datapoints show the ratios between the actual and expected numbers of points for (b) the upper prediction interval and (c) the lower prediction interval indicated on the x-axes, and the light gray shading shows the uncertainty of the model around the ratio of 1. The dashed datalines are identity lines, with no difference between the actual and expected numbers. Sample time optimization was performed using the WinPOPT library two-compartment model with first-order absorption. This is a simpler model than the final population pharmacokinetic model, adjusted to reflect the structure of the library model prior to performing the sample time optimization. The absorption process was simplified from the sequential zero- then first-order process to a first-order process only, and the IOV terms for D1, Frel, and ka were also removed. The actual parameter values used for the sample time optimization are presented in table IX. The simplified model retained the influence of dose on ka, thus the value for ka (0.403/hour) is that calculated for a 50 mg dose. The results of sample time optimization are shown in table X. Table IXGLPG0259 parameter estimates used for sample time optimization Table XGLPG0259 parameter estimates used for sample time optimization The gold-standard design (six samples per subject after both the 7th and 84th doses) criterion value was set at 100%. Further, the imprecision in the estimated CL/F value under this design was only 4.2%, indicating that the design was able to estimate CL/F well. The poor design (a single sample per subject after each of the 7th, 14th, 28th, 56th, and 84th doses, at 2 hours postdose) gave a criterion value that was 0.026% of that for the gold-standard design, and CL/F was estimated extremely imprecisely. Design no. 4, where a single sample was taken per subject but at different times per visit and always in the afternoon (thus at 5, 6, 7, 8, and 9 hours postdose across the visits) gave rise to a criterion ratio of 4.1%, and CL/F was estimated with 64.4% imprecision. Thus design no. 4 was not very good but was a considerable improvement over the poor design. Design no. 5 was similar to design no. 4, but samples were allowed to be taken both in the morning and in the afternoon, and the criterion ratio improved to 8.0% and CL/F was estimated with 22.1% imprecision. As can be seen in table IX, various designs were tested, but the greatest improvement came when the spread of the timing of the samples over the dosing interval was as wide as possible across the visits (design no. 8), and the criterion ratio was 25.8% and CL/F was estimated with 6.2% imprecision. Allowing more than one sample to be taken on one of the visits (design nos. 11 and 12) did not improve the criterion ratio or improve the precision with which CL/F was estimated, probably because a design with five samples per subjects was already adequate as a sparse sample design. BODY.DISCUSSION: After single and daily repeated administration, GLPG0259 was slowly absorbed and eliminated. On the basis of a statistical ANOVA, the exposure to GLPG0259 increased in proportion to the dose over a 30–150 mg single-dose range and a 25–75 mg repeated-dose range. In the population pharmacokinetic model developed with data from the three first phase I studies, the Frel for GLPG0259 increased with increasing dose, while the ka decreased with increasing dose up to 50 mg and was then reasonably constant. Conversely to the conclusion drawn from the ANOVA on dose-normalized parameters, these changes in Frel and ka detected during the development of the population pharmacokinetic model would be a sign of non–dose-proportional pharmacokinetics. It is not unusual to observe deviation from dose proportionality within a dose range as wide as 1.5–150 mg. In addition, a population approach is much more sensitive than standard statistical analysis for finding and characterizing dose non-linearity.[16] More data would be needed, especially at higher dose levels, to refine the model and the relation of ka and Frel to the dose to draw definitive conclusions on the dose linearity of GLPG0259 pharmacokinetics. The most frequently reported AEs following repeated administration with GLPG0259 were related to gastrointestinal disorders (loose stools, nausea, abdominal pain, or discomfort). These events, reported only at doses of 50 mg and higher, could be explained by the residence time of GLPG0259 in the gastrointestinal tract. Indeed in a whole-body autoradiography with [14C]-radiolabeled compound administered in a mouse model (3 mg/kg [14C]-GLPG0259), a huge amount of radioactivity was localized 4 and 8 hours postdose in the small and large intestine contents, as well as in the gallbladder, suggesting slow and incomplete absorption and/or intestinal secretion directly or via the bile (data not shown). Apart from gastrointestinal disorders, no systemic AEs were reported after repeated dosing with GLPG0259. Thus an increase in Frel with increasing dose should not be of concern as long as systemic exposure in humans remains below the 'no observed adverse effect level' (NOAEL) exposures in animal species. Methotrexate is an antifolate used with varying degrees of success in the treatment of cancer and autoimmune diseases.[17] Low-dose pulse methotrexate has emerged as the anchor drug in patients with RA because of its favorable risk-benefit profile.[18] Methotrexate is mainly eliminated by the kidney as intact drug, regardless of the route of administration. Glomerular filtration is the predominant pathway, with an additional active secretory process via organic anion transporters (OATs). Active biliary secretion also plays a role in methotrexate elimination, with variable amounts of methotrexate available for enterohepatic recirculation. Many drugs currently used in RA are known to interact with methotrexate pharmacokinetics: chloroquine reduces intestinal absorption; non-steroidal anti-inflammatory drugs can lead to a decrease in renal blood flow and glomerular filtration, and can compete with drug transporters for active renal tubular secretion; and calcium folinate has been shown to shorten the mean residence time of methotrexate in the kidney and liver.[15] GLPG0259 was eliminated by metabolism as well as renal excretion. Total body clearance of GLPG0259, predicted using allometric scaling of intravenous data from several animal species corrected for their maximum lifespan, as described by Mahmood,[19] was moderate, with a value of 54 L/h (data not shown). CLR determined in healthy subjects accounts for about 9 L/h of the total body clearance. As reported previously, the presence of radioactivity in the gallbladder after [14C]-GLPG0259 administration in a mouse model may suggest the elimination of GLPG0259 or metabolites via bile secretion and a possibility for re-absorption and enterohepatic recirculation. As GLPG0259 was intended to be developed for use as a monotherapy or in combination with drugs such as methotrexate, and taking into account the common routes of elimination of both methotrexate and GLPG0259, it was of interest to get preliminary information on the potential for drug-drug interaction between these two compounds at an early stage in drug development. Although this analysis was performed on a small subset of subjects (n = 6), no modification of the absorption or the elimination of methotrexate was noted after a daily dose of GLPG0259 50 mg. The t1/2,λz values for methotrexate estimated with and without GLPG0259 were about 3.4 and 3.1 hours, respectively. The range of boundary values for t1/2,λz reported in the literature is quite large (6–69 hours).[17] This variability may be partly related to differences in blood sampling between studies. The terminal log-linear phase cannot be determined accurately if the sampling interval is too short and/or too few blood samples are collected after 12 hours postdose.[15,17] Concerning GLPG0259, concomitant dosing with methotrexate had no impact on its bioavailability (Cmax and AUC24h). Although the GLPG0259 free-base oral solution showed good bioavailability, this formulation is not easy to handle in long-term trials. Consequently, development of an oral solid dosage form was envisaged. GLPG0259 free base is poorly soluble in aqueous media, and its solubility decreases with increasing pH (<0.01 mg/mL at pH 7). Two approaches were developed in parallel to overcome this low solubility and to improve compound bioavailability after dosing in a solid dosage form. The first approach was a salt screening, which resulted in the selection of the fumarate salt for further formulation development work. The water solubility of the GLPG0259 fumarate salt, as compared with that of the free base, was increased to 1.9–2.7 mg/mL. The impact of the improvement in solubility was confirmed in a comparative bioavailability study in fasted dogs. In that study, GLPG0259 fumarate salt (suspension in 20% [w/v] hydroxypropyl-ß–cyclodextrin, pH 3, or as crystalline powder in capsule form) resulted in plasma exposure similar to that of GLPG0259 free base in suspension in 20% acidified hydroxypropyl-ß–cyclodextrin, but 4-fold higher plasma exposure than that of GLPG0259 free-base crystalline powder in capsule form (data not shown). In humans, administration of GLPG0259 fumarate salt as a crystalline powder in capsule form leads to 50% lower bioavailability than that of GLPG0259 free base given as a solution in 40% (w/v) hydroxypropyl-ß–cyclodextrin, pH 3 (study 3). The lower performance of the fumarate capsule in humans than in dogs is explained by the higher percentage of hydroxypropyl-ß–cyclodextrin (40% versus 20%) in the liquid formulation, which enhances GLPG0259 free-base solubility. Concomitant food intake with the solid dosage form prevents this decrease in bioavailability by increasing the solubility further. The second approach was the improvement of GLPG0259 solubility by physical modifications of the drug substance – in particular, the development of solid dispersion formulations with GLPG0259 free base in an amorphous form homogenously dispersed in a polymer matrix. The free-base solid dispersion as a powder or pellets filled into capsules was tested in fasted dogs, and both solid dispersion formulations showed GLPG0259 plasma exposure similar to that of the fumarate salt as a crystalline powder in capsule form. Similar results were obtained in humans (study 4). In the Biopharmaceutical Classification System, drugs are classified according to measurements of solubility and permeability.[20] Regarding GLPG0259, it is a poorly soluble compound, with solubility that decreases with increased pH. The absorption of GLPG0259 was not measured in vivo in humans (there are no data after intravenous dosing), but its permeability assessed using the well established in vitro system, based on the human adenocarcinoma cell line Caco-2, was good, with an apparent permeability coefficient (Papp) of 12.4 10-6 cm/s and limited efflux (Papp B2A/Papp A2B = 2). Thus on the basis of these data, GLPG0259 could be classified as a class 2 compound, with in vivo drug dissolution then being the rate-limiting step for its absorption. Salt selection or solid dispersion development allows this issue to be overcome and increases the solubility and dissolution rate of GLPG0259, leading to an improvement in the bioavailability of the oral solid dosage forms to be used in future clinical trials. BODY.CONCLUSION: In summary, the investigation of safety/tolerability and pharmacokinetics in the early development phase showed that single and repeated doses of GLPG0259 were safe and well tolerated. The most common AE reported was mild gastrointestinal discomfort. The pharmacokinetics characterized in healthy male subjects showed no major obstacles and supports a once-daily oral regimen in patients.

TITLE: Prothrombin complex concentrate versus fresh frozen plasma in patients on oral anticoagulant therapy undergoing cardiac surgery: a randomized study ABSTRACT: BODY.INTRODUCTION: To reverse oral anticoagulant (OAC) therapy, a number of treatment modalities is available. Fresh frozen plasma (FFP) is effective and is currently used for coagulation factor replacement, carrying a risk of volume overload, transmission of infective agents and being time consuming. Variable and frequently low potency of clotting factors results in minor haemostatic effects compared with prothrombin complex concentrates (PCC), which are considered very effective and safe. PCC PPSB-SD® has constant, highly concentrated levels of factors II, VII, IX and X compared with FFP. We studied the efficacy of the intraoperative administration of PCC and FFP in patients on OAC therapy undergoing heart surgery with cardiopulmonary bypass (CPB). BODY.METHOD: After Ethical Committee approval and informed consent, 40 patients (P group, n = 20; FFP group, n = 20) with a preoperative INR ≥ 2.1 were studied. PCC was supplied as 500 IU factor IX (20 ml) vials. The dose was calculated on the basis of body weight, the initial INR and the target INR aiming at an INR of 1.5 after protamine. One-half of this dose was administered before the start of CPB. After weaning from CPB and protamine, the second half-dose was given in order to reach a postoperative INR ≤ 1.5. In case the INR value was still too high a further dose of PPSB was given. In the FFP group, each patient received 4 units: one-half of this dose was given before CPB and the other half after CPB. Additional FFP was given until the INR had reached a satisfactory level. In cases of poor response and/or if there was a danger of volume overload, PCC was given. A portable coagulation monitor (CoaguChek) was used for INR measurements. Blood sampling was preoperative (T-1), preincision (T0), preadministration and postadministration before CPB (T1, T2), during CPB at 15 and 45 min (T3, T4), at the end of CPB (T5), after protamine administration (T6), and 15 and 60 min and 3 and 16 hours post-CB (T7–T10). Analyses performed were: INR, PT, Hct, ACT, aPTT, ad factors II, VII, IX, X and FV. The amount of blood lost in the chest tube drainage and the blood products administered was also registered. Statistical evaluations were performed using the Student t test, repeated-measurements ANOVA and Fisher's exact test. BODY.RESULTS: The P group was more successful in reaching the target INR. In the FFP group 16/20 (80%) patients received an additional dose of PPSB vs 6/20 (30%) in the PCC group. The INR with PCC treatment dropped sooner below 1.5 than that in the FFP group. More patients in this group reached the target INR in the first hour after ending CPB (T7, P < 0.007). We found a significant difference between groups in factor II (P = 0.023) and factor X (P = 0.008) levels over time. BODY.CONCLUSION: The results of our study support the use of PCC in patients on OAC therapy facing semi-urgent or urgent cardiac surgery. Treatment with PCC reverses anticoagulation safely, more rapidly and more effectively than FFP.

TITLE: Effects of cranberry ( ABSTRACT.BACKGROUND: The aim of this study was to analyze the effect of supplementation with cranberry (Vaccinum macrocarpon) on the levels of pro-inflammatory cytokines, hepcidin and selected markers of iron metabolism in rowers subjected to exhaustive exercise. ABSTRACT.METHODS: This double-blind study included 16 members of the Polish Rowing Team. The subjects were randomly assigned to the supplemented group (n = 9), receiving 1200 mg of cranberry extract for 6 weeks, or to the placebo group (n = 7). The participants performed a 2000-m test on a rowing ergometer at the beginning and at the end of the preparatory camp. Blood samples were obtained from the antecubital vein prior to each exercise test, one minute after completing the test, and after a 24-h recovery period. The levels of hepcidin, interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), ferritin, iron, soluble transferrin receptor (sTfR) and myoglobin were determined, along with total iron-binding capacity (TIBC), unbound iron-binding capacity (UIBC) and total antioxidant capacity (TAC). ABSTRACT.RESULTS: Both prior and after the supplementation, a significant post-exercise increase in the concentration of IL-6 was observed in both groups. At the end of the study period, cranberry-supplemented athletes presented with significantly higher resting, post-exercise and post-recovery levels of TAC than the controls. However, a significant exercise-induced increase in the concentrations of TNF-alpha, myoglobin and hepcidin was observed solely in the control group. ABSTRACT.CONCLUSION: Supplementation with cranberry extract contributed to a significant strengthening of antioxidant potential in individuals exposed to strenuous physical exercise. However, supplementation did not exert direct effects on other analyzed parameters: inflammatory markers and indices of iron metabolism (TNF-alpha, hepcidin and myoglobin). BODY.BACKGROUND: Normal level of iron in the body is a key determinant of health [1]. Recent studies showed that concentration of iron may be affected by many factors, inter alia by strenuous and/or long-term physical exercise. The exercise with such characteristics induces systemic inflammation, which is reflected by enhanced release of hepcidin and resultant disorders of iron metabolism. One consequence of these unfavorable changes is hypoferremia, which may eventually lead to anemia [2, 3]. Some authors tried to prevent disorders of iron metabolism, correcting its deficiency by means of dietary supplementation or intravenous injection. Reardon [4] demonstrated that iron injections may increase skeletal muscle iron content in mice. According to these authors, the elevated level of iron induced oxidative stress and reduced exercise performance. Burden [5] used a single 500 mg intravenous iron injection in Fe-deficient non-anemic runners. While an improvement of iron metabolism parameters has been demonstrated four weeks post-injection, this was not reflected by better aerobic capacity. Similar changes were also observed following oral administration of iron [6, 7]. Altogether, this evidence suggests that although supply of exogenous iron may improve its metabolic parameters, this is not reflected by expected changes in physical capacity. Disorders of iron homeostasis are postulated to be linked to excessive generation of reactive oxygen species. Disruption of the pro-oxidant-antioxidant balance may be associated with the release of iron from active sites of proteins and resultant increase in labile iron pool (LIP). As a result, also concentration of iron ions that can take part in Fenton's reaction increases, which exacerbates oxidative stress on the one hand, and may promote systemic inflammation and hepcidin release on the other [8]. Administration of compounds with high antioxidant potential and strong iron-chelating activity seems to be a reasonable way to normalize iron metabolism and to attenuate systemic inflammation induced by strenuous physical exercise. Such compounds are present in many fruits and vegetables, including cranberry fruits [9, 10]. American cranberry (Vaccinium macrocarpon Ait.), also referred to as bearberry and large cranberry (Oxycoccus macrocarpos), is a member of Ericaceae family, which had been for a long time used in traditional folk medicine, inter alia as a treatment for urinary tract infections. Many recent clinical studies and in vitro experiments confirmed that some components of cranberry fruits exert beneficial effects in the urinary tract [11, 12] and may alleviate gastrointestinal ailments associated with Helicobacter pylori infection [13]. Also antiviral (against reoviruses, rotaviruses and influenza viruses), antioxidant and anti-inflammatory effects of these compounds were documented [14–17]. Available evidence suggests that for the reduction of free radicals and modulation of inflammatory response are responsible polyphenolic compounds present in cranberries, such as anthocyanins, flavonols, and flavanols [10]. Bioavailability of these compounds is determined by their metabolism and absorption. The study of healthy adults conducted by McKay et al. [18] revealed a considerable individual variability in the concentration of polyphenolic compounds and their metabolites after ingestion of low-calorie cranberry juice cocktail, as well as an increase in antioxidant capacity of blood plasma. The latter has been observed for up to 8 h post-ingestion [18], which may potentially constitute an important factor in control of oxidative stress. A review of available evidence demonstrates that previous research centered mainly around the effects of cranberry supplementation on the indices of oxidative stress and inflammatory markers in animals and humans. However, to the best of our knowledge, none of the studies analyzed if and to what extent the administration of cranberry products may modulate iron metabolism under conditions of enhanced oxidative stress and inflammation induced by strenuous physical exercise. The aim of this study was to analyze the effects of supplementation with large cranberry extract on antioxidant capacity, selected inflammatory markers and parameters of iron metabolism in individuals exposed to strenuous physical exercise; the latter is considered as a disruptor of systemic iron turnover. BODY.METHODS.STUDY POPULATION: The study included 16 male members of the Polish Rowing Team (13 heavyweight and 3 lightweight rowers). The basic characteristics and sport classes of the athletes are presented in Table 1. Inclusion and Exclusion criteria are listed in Table 2. The study was conducted between April and June, during a 6-week training camp taking place between the preparatory and competitive phase of a yearly training cycle.Table 1Basic characteristics of the studied groups (mean ± standard deviation)ParametersSupplemented group (n = 9)Control group (n = 7)Age (years)21.3 ± 0.8221.3 ± 0.71Body mass (kg)93.6 ± 6.0281.6 ± 9.44Body height (cm)192.6 ± 6.53189.0 ± 4.62Duration of training (years)7.5 ± 1.56.9 ± 1.2 P = NS for all between-group comparisons Table 2Inclusion and exclusion criteria for the study participantsTotal NSupplemented group NControl group NInclusion criteria • Male athletes qualified to Polish Youth Rowing Team (age 19–23 years), with physician-certified permission to participate in trainings and competition• 19• 10• 9Exclusion criteria• 3• 1• 2 • Medication intake (inflammatory drags, medications known to affect iron metabolism)• 2• 1• 1 • Injury excluding participation in trainings• 1• 0• 1 BODY.METHODS.FOOD INTAKE: Throughout the entire study period, the athletes resided and took their meals exclusively at one of the Olympic Games Training Centers. Their regular menu consisted of a mixed diet, providing recommended dietary allowance (RDA) of carbohydrates, proteins, fats, and micronutrients (vitamins and minerals), as stated in the Polish Nutrition Society guidelines [19]. The athletes' daily food, caloric, and fruit and vegetable intakes were constant throughout the study period. None of the study subjects presented with any health problems nor took any anti-inflammatory drugs, vitamins or medications that may interfere with iron metabolism. The participants were under no dietary restrictions. BODY.METHODS.EXPERIMENTAL PROCEDURE: The athletes who were randomized to the supplemented group (n = 9) received 720-mg capsules containing highly-concentrated large cranberry extract. Each capsule contained 360 mg of cranberry extract (including 36 mg proanthocyanidins), i.e. an equivalent to 4320 mg of cranberry fruits. Aside from cranberry extract, standardized for proanthocyanidin content (10%), each capsule contained also beef-pork gelatin, filler (cellulose), anti-caking agents (magnesium salts of fatty acids, talc) and colorants (titanium dioxide, azorubine, patent blue V). Capsules were manufactured by Synoptis Pharma Sp. Z o.o., Poland, and their expiry date was specified as March 2017. The athletes ingested two capsules with cranberry extract daily (one in the morning and one in the evening) for six weeks. The subjects from the control group (n = 7) were given dyed gelatin placebo capsules containing "Poznańska" flour (manufactured at Polskie Zakłady Zbożowe, Krakow) twice a day for 6 weeks, at the same time and with the same dosage regimen. BODY.METHODS.ROWING PERFORMANCE TEST: The athletes performed a controlled 2000-m time test on the first day (prior to the supplementation) and at the end of the training camp (after the supplementation). Each subject had to cover the distance on a rowing ergometer (Concept II, USA) in as short a time as possible. Because the results of both tests were taken into consideration during the selection to the championship team, the athletes were well motivated to perform both trials at a maximal effort. Each test was preceded by a 5-min individual warm-up. BODY.METHODS.SAMPLE TREATMENT: Blood samples were obtained before the 2000-m test (in the morning, after an overnight fast), 1 min after completing the test, and after a 24-h recovery period. Blood samples were collected to tubes without additives and following centrifugation (10 min at 3500 rpm), the sera were stored at −80 °C until analysis. Additionally, capillary blood samples were obtained from the earlobe before and after each exercise test to assess the athletes' lactic acid levels. BODY.METHODS.MEASUREMENTS: Serum IL-6 was measured using a commercially available enzyme-linked immunosorbent assay (ELISA; Quantikine HS, R&D Systems, Minneapolis, USA) with an assay range of 0.38-10 pg⋅ml−1. The intra assay and inter assay coefficients of variation (CVs) were 6.4 and 8.5%, respectively. Serum concentrations of TNF-alpha (in pg/ml) were quantified using a commercially available enzyme immunoassay (ELISA; Quantikine HS, R&D Systems, Minneapolis, USA) with an assay range of 0.5 to 32 pg⋅ml−1. The intra assay and inter assay CVs were 8.2 and 11.5%, respectively. Serum hepcidin was measured using a commercially available ELISA (Wuhan EIAab Science Co., China) with an assay range of 0.187-12 ng⋅ml−1. The intra assay and inter assay CVs were 5.2 and 7.9%, respectively. Iron concentration and TIBC were measured with the colorimetric method with chromogens (BioMaxima S.A., Poland); the results were expressed in μg/dL. Unsaturated iron-binding capacity (UIBC) was calculated from the formula: UIBC = TIBC – Fe. Myoglobin concentration was determined immunochemically, with an aid of the Myoglobin ELISA kit (BioCheck, USA); the results were expressed in ng⋅ml−1. The intra assay and inter assay CVs were 3.4 and 5.5%, respectively. Serum ferritin levels were determined immunochemically, with an aid of a commercially available diagnostic kit (Demeditec, Germany); the results were expressed in ng⋅ml−1. The intra assay and inter assay CVs were 4.6 and 6.0%, respectively. Concentrations of soluble transferrin receptor (sTfR) were determined immunochemically with a commercially available diagnostic kit (BioVendor, Czech Republic). The intra assay and inter assay CVs were 8.4 and 13.5%, respectively. Total antioxidant capacity (TAC), considered a marker of plasma antioxidant capacity, was assessed with a commercially available kit (Cayman, USA); the results were expressed as mmol⋅l−1. The intra assay and inter assay CVs were 3.3 and 5.7%, respectively. Concentration of lactate (La) in capillary blood was determined immediately after sampling, using a commercially available kit (Dr Lange, Germany); Lactate concentrations were expressed in mmol⋅l−1. If necessary, the results were adjusted for hemoconcentration, using the exercise-induced changes in hematocrit as a covariate. BODY.METHODS.STATISTICAL ANALYSIS: Statistical analyses were performed with STATISTICA v. 10.0 software package (StatSoft, Cracow, Poland). All parameters were compared using 2 (supplemented and placebo group) × 3 (timing of measurement) repeated measures analysis of variance (ANOVA). Normal distribution of the data was verified with Shapiro-Wilk test. Whenever significant changes were documented on ANOVA, Fisher's post-hoc tests were conducted to identify the source of significant differences. Student's unpaired t-test was used to compare the study groups in terms of their anthropometric characteristics. Except for the rowing time, the results of the 2000-m tests performed prior to and after the supplementation were subjected to intragroup comparisons with Student's paired t-test, and for intergroup comparisons with Student's unpaired t-test. The results are presented as arithmetic means ± standard deviations (SD). The threshold of statistical significance for all tests was set at p < 0.05. BODY.RESULTS: The subjects from the supplemented group and the controls did not differ significantly in terms of their mean age, body height, body weight, and years of training (Table 1). No significant intergroup differences were found in mean power output and total row time during the 2000-m test performed at the beginning of the training camp. Furthermore, no significant differences in the pre- and post-test blood lactate levels were documented when the results for Trial I were compared to those of Trial II (Table 3).Table 3Changes in 2000 m rowing ergometer performance before and after supplementationParametersSupplemented group (n = 9)Control group (n = 7)BeforeAfterBeforeAfterPower (watt)452 ± 15.6457 ± 12.5406 ± 36.6417 ± 39.5(W x kg−1)4.84 ± 0.254.88 ± 0.284.99 ± 0.285.03 ± 0.43LAmin (mmol x L−1)a 1.8 ± 0.231.8 ± 0.191.8 ± 0.261.9 ± 0.18LAmax (mmol x L−1)a 14.2 ± 2.9115.0 ± 2.0915.6 ± 2.1416.8 ± 1.44Time (s)366.7 ± 4.39365.3 ± 3.24380.8 ± 11.34377.2 ± 11.86Values represent the mean ± standard deviation. There were no significant differences after supplementation relative to before supplementation (P < 0.05) a LA lactate acid Values of total antioxidant capacity (TAC) determined at Trial I and II are shown on Fig. 1. This parameter turned out to be modulated both by physical exercise (p < 0.001) and cranberry supplementation. Prior to the supplementation (Trial I), both groups of athletes showed a significant post-exercise decrease in serum TAC. At trial II, cranberry-supplemented athletes presented with significantly higher resting, post-exercise and post-recovery levels of TAC than the controls. These were the athletes from the supplemented group, however, who showed a significant post-exercise decrease in serum TAC at Trial II.Fig. 1Total antioxidant capacity (TAC) levels during exercise tests performed before and after the supplementation (mean ± SD). Note. □ - SUPL = supplemented group; − PLA = placebo group; B = baseline; Ex = immediately after the exercise; R = after a 1-day recovery; * - significantly different compared to PLA group; † − significantly different compared to baseline level; # -significantly different compared to post-exercise level Changes in serum levels of IL-6 observed prior to and after cranberry supplementation are shown on Fig. 2a. A significant effect of physical exercise on IL-6 concentration (main effect, p < 0.001) has been documented on ANOVA. At both Trial I and II, a significant post-exercise increase in the concentration of this cytokine was observed in either group, with subsequent normalization at the pre-exercise level following a 24-h recovery. Supplementation with cranberry extract did not exert a significant effect on IL-6 concentration (main effect, p = 0.051).Fig. 2Changes interleukin 6 (a) and tumor necrosis factor alpha (b) levels during exercise tests performed before and after the supplementation (mean ± SD). Note. IL-6 = interleukin 6; TNF α = tumor necrosis factor alpha; □ − SUPL = supplemented group; − PLA = placebo group; B = baseline; Ex = immediately after the exercise; R = after a 1-day recovery; † − significantly different compared to baseline level; # -significantly different compared to post-exercise level Exercise-induced changes in TNF-alpha levels are presented on Fig. 2b. Strenuous physical exercise induced statistically significant changes in this parameter, as confirmed on ANOVA (main effect, p < 0.001). While no statistically significant differences between pre-exercise, post-exercise and post-recovery values of this parameter were found in both the cranberry-supplemented athletes and the controls at Trial I, athletes from the latter group showed a significant exercise-induced increase in serum concentration of TNF-alpha at Trial II. Based on the results of ANOVA, cranberry supplementation did not exert a statistically significant effect on TNF-alpha levels (main effect, p = 0.118). At trial II, a significant post-exercise increase in hepcidin level was observed in the controls but not in the cranberry-supplemented athletes. However, no significant effect of cranberry supplementation on this parameter was demonstrated on ANOVA (main effect, p = 0.177; Fig. 3a).Fig. 3Changes in serum levels of hepcidin (a), iron (b), and myoglobin (c) levels during exercise tests performed before and after the supplementation (mean ± SD). Note. □ - SUPL = supplemented group; − PLA = placebo group; B = baseline; Ex = immediately after the exercise; R = after a 1-day recovery; † − significantly different compared to baseline level Supplementation with cranberry extract did not exert a significant effect on serum iron (main effect, p = 0.516). However, a significant post-exercise increase in serum concentrations of iron was documented in both the cranberry-supplemented athletes and the controls at Trial II (Fig. 3b). Serum concentration of myoglobin was modulated solely by physical exercise (main effect, p < 0.001). While no significant exercise-induced changes in this parameter were observed in both groups at Trial I, a significant increase in serum concentration of myoglobin was documented in the controls at Trial II (Fig. 3c). Irrespective of the group, statistically significant exercise-induced changes in TIBC (Fig. 4a) and UIBC (Fig. 4b) were observed at Trial II, but not at Trial I. Both parameters increased in response to physical exercise and then returned to their pre-exercise levels following the recovery period. Based on the results of ANOVA, cranberry supplementation did not exert a significant effect on any of these parameters.Fig. 4Changes TIBC (a), UIBC (b), ferritin (c) and sTfR (d) levels during exercise tests performed before and after the supplementation (mean ± SD). Note. TIBC = total iron-binding capacity; UIBC = unsaturated iron-binding capacity; sTfR = soluble transferrin receptor - □ SUPL = supplemented group; − PLA = placebo group; B = baseline; Ex = immediately after the exercise; R = after a 1-day recovery; † − significantly different compared to baseline level; # -significantly different compared to post-exercise level The data on sTfR and transferrin levels are presented on Fig. 4 (c and d). Neither cranberry supplementation nor physical exercise exerted statistically significant effects on these parameters. BODY.DISCUSSION: The aim of our study was to analyze the effects of large cranberry intake on antioxidant capacity, selected parameters of iron metabolism and inflammatory markers in athletes exposed to strenuous physical exercise. The study showed that physical exercise contributed to a significant decrease in serum TAC (Fig. 1). Similar findings were previously reported by Finaudi [20] (2006) and Margonis [21], according to whom pre-competitive and competitive phases of a yearly training cycle are associated with enhanced oxidative stress and a decrease in antioxidant capacity. Post-exercise decrease in TAC may result in accumulation of inadequately deactivated free radicals that may initiate peroxidation of polyunsaturated fatty acids of erythrocyte membranes [22]. Fiorani [23] demonstrated that human erythrocytes can uptake flavonoids from extracellular milieu via a passive diffusion mechanism, becoming a natural reservoir of these compounds. While most flavonoids (up to 85%) are found in the cytosolic fraction, some are bound to plasma membranes. Arora [24] and Terao [25] showed that flavonoids, similar to cholesterol and alpha-tocopherol, are localized near the surface of the membrane, between phospholipid bilayer and aqueous phase. Such location of flavonoids is crucial for stabilization of the membrane, making it less prone to oxidation due to a decrease in its fluidity [26]. Our findings imply that administration of cranberry extract may stimulate an increase in serum TAC (Fig. 1). Compared to the controls, cranberry-supplemented athletes presented with significantly higher levels of TAC at rest and following a 24-h recovery. Also other authors observed an increase in antioxidant potential after cranberry supplementation [27, 28]. According to Basu [29], administration of cranberry juice (480 ml/day for 8 weeks) contributed to a significant increase in plasma antioxidant capacity, as well as to a decrease in oxidized LDL and malondialdehyde levels in individuals diagnosed with metabolic syndrome. Similar results were also documented in a group of healthy men who consumed cranberry juice (7 ml/kg body weight per day) for 14 days [30]. Celik [31] showed that antioxidant capacity of cranberry fruits is greater when they accumulate more anthocyanins. Probably, this association can be explained by previously documented ability of anthocyanins to bind toxic iron ions [32]. Physical exercise, especially strenuous, induces an array of unfavorable changes, such as a shift in the pro-oxidant-antioxidant balance toward oxidation, hyperthermia, metabolic acidosis, hypoglycemia and hemoconcentration. All of them may contribute to a decrease in the osmotic resistance of erythrocytes and make them more susceptible to hemolysis [33]. Enhanced hemolysis may in turn lead to a dramatic increase in circulating pool of redox-active iron. Fenton's reaction, a consequence of increased availability of Fe, constitutes the principal mechanism of iron toxicity. High reactivity and low specificity of •OH substantially hinder protection of cell components and body fluids against the toxic effects of iron. After being released from cells, iron may be bound by ferritin or low-molecular-weight cytoplasmic ligands, forming the so-called labile iron pool (LIP). As a crossroad of cell iron metabolism, cytoplasmic LIP undergoes dynamic changes. LIP is built of iron that has been delivered to the cell or recovered from degraded intracellular Fe-binding proteins, and serves as a source of this metal for synthesis of heme and iron-sulfur clusters. Kvam [34] demonstrated that iron released during enzymatic degradation of heme contributes to a rapid increase in the LIP; according to these authors, this may result in a transient (2- to 3-h) increase in the susceptibility of erythrocytes to UVA-induced oxidative stress. Both cranberry supplemented athletes and controls participating in our study showed a significant post-exercise increase in serum iron (Fig. 3b), TIBC and UIBC at Trial II (Fig. 4a and b). Elevated serum levels of iron not only may trigger free radical-mediated processes, but also activate immune system and induce inflammation as a form of an early protective response [35]. Available evidence suggests that some constituents of cranberry fruits may act as immunostimulants, mitigating the inflammation [9]. Supplementation with cranberry extract did not alter significantly the levels of inflammatory markers, IL-6 and TNF-alpha, in our athletes. Statistically significant changes in these parameters were observed after exposure of the study subjects to strenuous physical exercise (Fig. 2 a and b). The exercise-induced increase in IL-6 level was observed irrespective of the group, at both Trial I and II. Then, following the 24-h recovery, IL-6 returned to its baseline level (Fig. 2a). While serum levels of TNF-alpha in athletes from both groups did not undergo significant changes at Trial I, a significant post-exercise increase in this parameter was observed in the controls at Trial II (Fig. 2b). According to Rämson [36], high-volume training stimulated an increase in plasma TNF-alpha concentration in male rowers. Similar results were also obtained in a study of mice subjected to a high-intensity physical training [37]. These findings suggest that a post-exercise increase in TNF-alpha level may be a useful marker of exercise load accumulation and predict the onset of possible overreaching/overtraining in athletes. Noticeably, we did not observe a post-exercise increase in the serum concentration of TNF-alpha in cranberry-supplemented athletes, although they were exposed to exactly the same exercise regimen as the controls. Anthocyanins, which are inter alia present in cranberry fruits, exert anti-inflammatory effect due to their ability to inhibit principal enzymes activated during the course of inflammation, i.e. cyclooxygenase 2 (COX-2) and lipoxygenase. Due to such properties, anthocyanins may block synthesis of inflammatory mediators, including prostaglandin E. A study using Caco-2/15 cells demonstrated that cranberry extract may prevent iron/ascorbate-mediated lipid peroxidation and counteract lipopolysaccharide-mediated inflammation, as evidenced by the decrease in pro-inflammatory cytokines: TNF-alpha, IL-6, cyclooxygenase-2 and prostaglandin E2 [15]. However, in a study of human subjects with metabolic syndrome, 60-day supplementation with cranberry juice did not cause significant changes in the levels of pro-inflammatory cytokines: TNF-alpha, IL-1 and IL-6, despite an improvement of some cardiovascular risk factors [38]. Immediately after the exercise, our rowers from the control group presented not only with the increase in TNF-alpha concentrations, but also with elevated levels of hepcidin (Fig. 3a) and myoglobin (Fig. 3c). Elevated level of myoglobin after exercise is a marker of muscle injury, whereas the increase in hepcidin is typically associated with disruption of iron metabolism [2]. Previous studies showed that the release of hepcidin is modulated by the availability of iron [39], inflammation [3], hypoxia [40] and changes in erythropoietic activity [41]. All these factors are also associated with strenuous physical exercise. Burden [5] showed that availability of iron is a key regulator of hepcidin level in elite athletes. In their study, a single intravenous injection of iron stimulated an increase in hepcidin level in iron-deficient non-anemic runners; this effect was independent of changes in IL-6 concentration. This suggests that the lack of post-exercise changes in TNF-alpha, myoglobin and hepcidin levels in supplemented athletes might reflect indirect beneficial effects of cranberry extract components: stimulation of antioxidant capacity and reduction of iron availability due to chelation [42]. Neither strenuous physical exercise no cranberry intake exerted a significant effect on the remaining parameters of iron metabolism in our rowers, i.e. sTfR and ferritin levels (Fig. 4c and d). This observation is consistent with the results published by other authors [5, 43–45]. BODY.CONCLUSION: Supplementation with cranberry extract contributed to a significant strengthening of antioxidant potential in individuals exposed to strenuous physical exercise. However, supplementation did not exert direct effects on other analyzed parameters: inflammatory markers and indices of iron metabolism (TNF-alpha, hepcidin and myoglobin). The lack of exercise-induced changes in inflammatory markers and parameters of iron metabolism seems to be indirectly linked to the enhancement of antioxidant potential. However, verification of this hypothesis requires further research on the mechanisms through which various components of cranberry extract, especially anthocyanins, may interfere with iron metabolic parameters. Furthermore, there is a need for specific guidelines regarding form, timing, frequency and dosage of cranberry supplements.

TITLE: Twelve-Month Follow-Up of a Randomized Controlled Trial of Internet-Based Guided Self-Help for Parents of Children on Cancer Treatment ABSTRACT.BACKGROUND: A substantial proportion of parents of children on cancer treatment report psychological distress such as symptoms of post-traumatic stress (PTSS), depression, and anxiety. During their child's treatment many parents also experience an economic burden. ABSTRACT.OBJECTIVE: The aim of this study was to evaluate the long-term efficacy of Internet-based guided self-help for parents of children on cancer treatment. ABSTRACT.METHODS: This study was a parallel randomized controlled trial comparing a 10-week Internet-based guided self-help program, including weekly support from a therapist via encrypted email, with a wait-list control condition. The intervention was based on cognitive behavior therapy (CBT) and focused on psychoeducation and skills to cope with difficult thoughts and feelings. Primary outcome was self-reported PTSS. Secondary outcomes were self-reported symptoms of depression, anxiety, health care consumption, and sick leave during the past month. Outcomes were assessed pre- and postintervention and at 12-month follow-up. Parents of children on cancer treatment were invited by health care personnel at pediatric oncology centers, and parents meeting the modified symptom criteria on the PCL-C were included in the study. Self-report assessments were provided on the Web. ABSTRACT.RESULTS: A total of 58 parents of children on cancer treatment (median months since diagnosis=3) were included in the study (intervention n=31 and control n=27). A total of 18 participants completed the intervention, and 16 participants in each group participated in the 12-month follow-up. Intention-to-treat analyses revealed significant effects in favor of the intervention on the primary outcome PTSS, with large between-group effect sizes at postassessment (d=0.89; 95% CI 0.35-1.43) and at 12-month follow-up (d=0.78; 95% CI 0.25-1.32). Significant effects in favor of the intervention on the secondary outcomes depression and anxiety were also observed. However, there was no evidence for intervention efficacy on health care consumption or sick leave. ABSTRACT.CONCLUSIONS: Using the Internet to provide psychological interventions shows promise as an effective mode of delivery for parents reporting an increased level of PTSS and who consider Internet-based interventions as a viable option. Future research should corroborate these findings and also develop and evaluate interventions and policies that may help ameliorate the economic burden that parents may face during their child's treatment for cancer. BODY.INTRODUCTION: Being a parent of a child diagnosed with cancer is burdensome. During the child's treatment, parents have to cope with the disease itself, invasive treatments, and uncertainty about the child's health and outcome. Indeed, parents of children on cancer treatment report psychological distress such as symptoms of post-traumatic stress disorder (PTSS) [1-3] and depression [4]. In addition, many parents of children on cancer treatment are affected economically. Cross-sectional studies indicate that the strain on household economy is highest during the first 6 months of treatment [5,6]. A recent longitudinal study found that the reduction in parents' working hours is highest 2 months after the child's diagnosis and then almost restored 1 year after end of treatment. However, 1 year after end of treatment, more mothers are still on sick leave than at the time of diagnosis [7]. Recent evidence from the same cohort suggests that reductions in working hours are restored 5 years after end of treatment [8]. There is evidence that psychological interventions can be of benefit to parents of children with chronic illnesses [9]. To the best of our knowledge, the intervention with best empirical support for reducing psychological distress among parents of children on cancer treatment is problem-solving training administered face to face [10,11]. This intervention is effective in reducing PTSS and depression among mothers of children on cancer treatment when compared with an inactive condition [11] and when compared with an active [12] condition consisting of nondirective support including active listening and reflective support. The intervention is 8 weeks in duration and has been evaluated up to 3 months after end of the intervention. A limitation of these studies is the lack of long term follow-up assessments. Importantly, there are no published findings regarding the effects of psychosocial interventions on health-related costs among parents of children on cancer treatment. As the medical treatment for pediatric cancer is highly specialized, families where a child is treated for cancer often live far from the center where the child receives its care. This distance can make it difficult to maintain proper psychosocial and psychological support. Research has reported that less than half of parents who report a need to see a psychologist have had the opportunity to do so [13]. Cognitive behavior therapy (CBT) provided via the Internet is a promising treatment modality for a range of conditions [14], including parents of children with traumatic brain injury [15]. Providing interventions via the Internet could potentially increase access of support for parents of children who are receiving treatment for cancer. Recent developments and ongoing work in the wider field of interventions for parents of children with cancer also include Web-based CBT to improve quality of life in families of young cancer survivors [16]. We have developed a 10-week guided self-help intervention for parents of children on cancer treatment to be administrated via the Internet [17]. The intervention is based on principles from cognitive and behavioral therapies and aims to teach parents skills to cope with distress related to their child's disease and treatment. We have previously reported that the intervention seems effective in the short term with significant reductions in the primary outcome PTSS and the secondary outcomes depression and anxiety, with large effect sizes at postassessment compared with a wait-list control condition [18]. The purpose of this study is to investigate the efficacy of the intervention including data from the controlled follow-up 12 months after randomization. PTSS was the primary outcome, and secondary outcomes included symptoms of depression, anxiety, and health-economic outcomes such as health care consumption and sick leave. BODY.METHODS.DESIGN: This is a parallel randomized controlled trial including pre- and postassessments and a controlled follow-up 12 months after randomization, comparing an Internet-based guided self-help program with a wait-list control condition. Participants were recruited consecutively from five of the six Swedish pediatric oncology centers. Participants allocated to the intervention condition received the intervention immediately after randomization, whereas participants allocated to the wait-list condition received the intervention 12 months after randomization. Neither participants nor therapists in the study were blind to condition allocation. This study relied on self-reported outcomes. BODY.METHODS.PARTICIPANTS AND PROCEDURE: Participants and procedure have been described in detail previously [18]. In brief, eligible participants were parents of children on treatment for a cancer disease who were fluent in Swedish, had access to a computer with an Internet connection, fulfilled the modified symptom criteria on the PTSD-Checklist-Civilian version (PCL-C) [19], a self-report instrument corresponding to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition (DSM-IV) criteria for PTSD [20], and did not suffer from any other psychiatric disorder in immediate need of treatment. The modified symptom criteria comprise scoring ≥3 on at least one of five symptoms of reexperiencing, one of seven symptoms of avoidance and numbing, and one of five symptoms of hyperarousal, corresponding to partial PTSD [21]. A power analysis indicated that a total of 72 participants needed to be included to, with a power of .80, detect a large effect size (d=0.80) on the PCL-C assuming P<.05. Given that data on health care visits and sick leave were collected and that such variables generally vary more than clinical efficacy, a sample of 120 participants was estimated appropriate. However, the participation rate during the 4 years of inclusion was considerably lower than expected, and due to administrative reasons inclusion had to be terminated before this sample size was reached. Potential participants were approached in person by a nurse or physician on the wards at the pediatric oncology centers 4-12 weeks after the child's diagnosis. In the initial protocol, potential participants were to be approached 1-2 weeks after diagnosis. However, during the first months of inclusion it was evident that this was not feasible, and parents often were approached later, and the protocol was changed to the time frame reported. Potential participants were provided written and oral information about the study and were asked for written consent to participate. Nurses responsible for the recruitment at each center were not affiliated with the research group responsible for the study but were reimbursed for their work. Parents were informed that the intervention would be 10 weeks in duration and require approximately 4 hours of work per week to complete. A psychologist from the research group contacted consenting parents via telephone, and parents were instructed to complete the screening and preassessment on the Web. Thereafter, a clinical interview with a psychologist was conducted via telephone. Three master's level psychologists conducted the interviews. Participants in the intervention condition completed the postassessment on the Web immediately after the intervention. Participants in the wait-list control condition completed the postassessment on the Web after the corresponding time (ie, 10-weeks post randomization). Participants in both conditions completed the follow-up on the Web 12 months after randomization. Thereafter, participants in the wait-list condition were offered access to the intervention. The procedure was approved by the regional ethics review board in Uppsala (Dnr 2008/238), and all participants provided written informed consent. Inclusion to the study started in April 2010. During the planning of this study, trial registration was less common in the field of psychology than it is currently. Therefore, this trial was not registered in a World Health Organization (WHO) accredited trial registry. BODY.METHODS.INTERVENTION: The intervention consists of Internet-based guided self-help provided during 10 weeks. The material has been described in detail [17] as well as its use in the current trial [18]. It consists of approximately 100 pages (A4 format) of text and visual material presented in nine modules. The intervention is based on CBT-principles [22-24] and focuses on psycho-education and teaching strategies to manage the current situation of being a parent of a child on cancer treatment and the stressors it entails. Components include relaxation training, coping with distressing thoughts and feelings, behavioral experiments, problem-solving, structured emotional writing, values and goal setting, general self-care, and maintenance of behavior change. Participants accessed the intervention material via a Web-based portal and were instructed to work with each module for 1 week. Each participant was assigned a therapist and was instructed to send completed homework assignments via the portal to the therapist once each week. The therapist provided written feedback on each assignment and general progress through the intervention via the portal. The sequence of modules was fixed, which enhanced treatment integrity. If participants had not submitted their homework they were sent an email reminder to log in to the system. During the recruitment phase and in the informed consent, participants had been informed that the intervention would imply about 4 hours of work per week. There were three therapists in the study. One licensed psychologist and two psychologists with a master's degree in psychology. The two nonlicensed psychologists received supervision from the licensed psychologist. The therapists were affiliated with the research group responsible for the study and independent from centers from which participants were recruited. Logging of therapist time and activities was not supported by the portal, but therapists were instructed to spend 15-20 min per week when providing feedback to each participant. Individuals randomized to the intervention who had their partner included in the study received individual feedback but were encouraged to work together with their partner during the intervention if that suited them. All participants were free to receive psychosocial services from the regular health care. These may have differed between centers as there are no standardized psychosocial services for parents within the Swedish pediatric oncology care setting. BODY.METHODS.OUTCOMES.PRIMARY OUTCOME: Post-traumatic stress symptoms related to the child's cancer disease were assessed with the PCL-C [19]. The PCL-C consists of 17 items rated on a 5-point scale (1=not at all and 5=extremely), corresponding to the items assessing the B (reexperiencing), C (avoidance and numbing), and D (hyper-arousal) criteria in the DSM-IV. The instrument has adequate internal consistency, test-retest reliability, and evidence for convergent and discriminant validity when compared with other well-established measures of PTSS, depression, and general anxiety [25]. A score of 44 or above on the full scale suggests a diagnosis of PTSD [26]. Cronbach alpha in the current sample was .84 at preassessment. BODY.METHODS.OUTCOMES.SECONDARY OUTCOMES: Depression was assessed with the Beck Depression Inventory-II (BDI-II) [27] consisting of 21 items rated on a 4-point scale (0-3). The BDI-II has good concurrent validity with the BDI and the Hamilton Psychiatric Rating Scale; the suggested cut-offs are 0-13 indicating minimal, 14-19 mild, 20-28 moderate, and 29-63 severe depression. Cronbach alpha in the current sample was .82 at preassessment. Anxiety was assessed with the Beck Anxiety Inventory (BAI) [28] consisting of 21 items rated on a 4-point scale (0-3). The BAI has good test-retest reliability and convergent validity; suggested cut-offs are 0-7 indicating minimal, 8-15 mild, 16-23 moderate, and 24-63 severe anxiety. Cronbach alpha in the current sample was .87 at preassessment. Economic outcomes were assessed with questions from the Trimbos iMTA questionnaire for costs associated with psychiatric illness (TiC-P) [29]. Questions used in this report assessed frequency of health care use and sick leave during the last month. For health care use, one or more visits to different health care providers were coded as a "yes." Similarly, for sick leave, 1 day or more of absence due to sickness during the last month was coded as a "yes." BODY.METHODS.RANDOMIZATION: Randomization was performed by a consultant independent from the research group. Proc Plan SAS version 9.1 (SAS Institute, Cary, North Carolina, USA) was used to generate the randomization schedule, and sealed envelopes were prepared by the consultant and handed to the research group. Parents were randomized in 1:1 ratio to intervention or wait-list, and the randomization was stratified according to center, parental gender, and whether a participant had a partner in the study or not. Partners were randomized to the same condition. BODY.METHODS.STATISTICAL ANALYSES: Independent samples t test, Mann Whitney U test, chi-square test, and Fisher exact test were used to test for between-group differences on demographic characteristics and outcomes at preassessment. For the continuous outcomes (PCL-C, BDI-II, and BAI), mixed effects modeling with full maximum likelihood estimation was used to examine potential change across assessments and effects of the intervention [30], including random intercepts and slopes. Analyses were conducted on the intention-to-treat (ITT) principle where all randomized participants are included in the analyses, assuming missing data to be missing at random [31]. The data missing mechanism was assessed before the main analyses by exploring the relationships between characteristics at preassessment and missing data. Condition was dummy coded with intervention=1 and control=0. Assessment (pre, post, and 12-month follow-up) was included as a continuous time-variable coded as pre=0, post=1, and follow-up=2. Models were tested stepwise with increasing complexity and selected based on model fit indices, that is, -2loglikelhood difference test, Akaike Information Criteria (AIC), and Bayesian Information Criteria (BIC). All models tested included variance component as covariance structure. The complete model development is presented in Multimedia Appendices 1-3. For each outcome, we started with an unconditional model including a random intercept and slope (model A), as a second step we added linear growth as a fixed effect (model B), as a third step we added group as a fixed effect (model C), and as a fourth step we added quadratic growth as a fixed effect (model D). Standardized effect sizes (Cohen d) between groups at postassessment and 12-month follow-up were calculated using the model estimated mean differences (by recoding the continuous time-variable to −1 0 1 to set the intercept at the postassessment and −2 −1 0 to set the intercept at the 12-month follow-up assessment) and standard deviations from preassessment [32]. The magnitude of the effect expressed in d was interpreted according to Cohen [33], that is, 0.2=small, 0.5=medium, and 0.8=large. Variables pertaining to economic data consisted of frequencies of health care visits and days on sick leave. Due to the distribution of these frequencies, these variables were recoded to binary categorical variables representing no visits or days on sick leave (coded as 0) and any one or more visits or days on sick leave (coded as 1). However, ITT analyses with these data using, for example, generalized estimating equations, were not feasible due to the small sample size, and results for this secondary outcome are based on the available data excluding participants with missing data. Between-group differences at pre-, post- and follow-up assessments were analyzed with chi-square tests or Fisher exact test. Due to the small sample size, clustering by center and child was not addressed in any of the analyses. Analyses were performed in IBM SPSS Statistics 22 (IBM Corporation, Armonk, New York, USA). BODY.RESULTS.RECRUITMENT AND BASELINE CHARACTERISTICS: Participant flow through the study is outlined in Figure 1. From April 2010 to May 2014, 747 potential participants were informed about the study and asked for consent to be contacted again of which 553 declined. A total of 174 were reached by telephone, and 92 of these completed the screening, preassessment and clinical interview, and were assessed for eligibility. Fifty-eight parents of 46 children were included and randomized. Baseline characteristics have been described previously [18], and observed characteristics are presented in Tables 1 and 2. There were no differences in baseline characteristics between groups except for the BAI with a higher score in the intervention group. The last follow-up assessment took place in August 2015. BODY.RESULTS.ATTRITION AND ADHERENCE: Fourteen participants in the intervention group (45%, 14/31) and 7 in the wait-list group (26%, 7/27) did not provide postassessments. Furthermore, 1 participant in the intervention group and 4 in the wait-list group did not provide follow-up assessments, resulting in 16 participants in each group at 12-month follow-up. At preassessment, there were no differences in terms of demographic characteristics or outcome measures between those who provided post- and follow-up assessments and those who did not (P value ranging .14-.98) except for gender where fathers were less likely to provide data at 12-month follow-up compared with mothers (χ21=6.4, P=.01). As reported previously [18], adherence to the intervention was operationalized as the numbers of treatment modules accessed and log-ins to the Web-based portal. In the intervention group, 6 participants did not start the intervention, and 7 discontinued before completion. A total of 18 out of 31 participants were considered as completers, representing 58% of those allocated to the intervention. For the ITT-sample, the median number (interquartile range [IQR]) of accessed modules was 4 (4), and the median number (IQR) of log-ins was 13 (22). For the completer-sample, the median number (IQR) of accessed modules was 5 (3.5), and the median number (IQR) of log-ins was 20 (20). Figure 1Consort diagram of participant flow through the study. Table 1 Baseline characteristics of parents. Characteristics Total sample n=58 Intervention n=31 Wait-list n=27 P value Mothers, n (%) 39 (67) 21 (68) 18 (67) .76 Partner also included in study, n (%) 22 (38) 12 (39) 10 (37) .90 Age, mean (SD a ) 38 (7.2) b 40 (7.4) c 36 (6.6) d .06 Living with child’s biological parent, n (%) 51 (88) 26 (84) 25 (93) .31 Completed university studies, n (%) 30 (52) 17 (55) 13 (48) .61 In active employment, n (%) 47 (81) 26 (84) 21 (78) .56 Median distance in km to POC e (IQR f ) 35 (61) 39 (62) 20 (50) .39 Experience of previous traumatic event(s), n (%) 26 (45) 14 (45) 12 (44) .96 Median months since child´s dx g (IQR) 3.0 (3.0) h 3.0 (2.25) i 3.0 (3.0) .39 Outcomes, mean (SD) PCL-C j 49.1 (10.3) 51.5 (9.4) 46.6 (10.7) .06 BDI-II k 20.6 (7.5) 21.6 (8.1) 19.3 (6.7) .24 BAI l 14.7 (7.6) 17.2 (7.8) 11.9 (6.3) <.01 a SD: standard deviation. b n=55. c n=29. d n=26. e POC: pediatric oncology center. f IQR: interquartile range. g dx: diagnosis. h n=57. i n=30. j PCL-C: PTSD-Checklist-Civilian version. k BDI-II: Beck Depression Inventory-II. l BAI: Beck Anxiety Inventory. Table 2 Baseline characteristics of children. Characteristics Total sample n=46 Intervention n=25 Wait-list n=21 P value Female, n (%) 25 (54) 16 (64) 9 (43) .15 Median age (IQR a ) 5 (9.0) 6 (10.5) 4 (8.0) .26 Diagnosis, n (%) .33 Leukemia 24 (52) 13 (52) 11 (52) Sarcoma 8 (17) 5 (20) 3 (14) Lymphoma 3 (7) 3 (12) 0 (0) CNS b -tumor 7 (15) 2 (8) 5 (24) Other malignant disease 4 (9) 2 (8) 2 (10) a IQR: interquartile range. b CNS: central nervous system. Table 3 Estimated outcomes of mixed effects models and effect sizes (n=58 in intention-to-treat analyses). Cohen d is the standardized mean difference and was calculated using the estimated mean difference and the standard deviation of the complete sample at the preassessment. Outcome Degrees of freedom Estimate (SE a ) 95% CI F test value t test value P value PCL-C b Intercept 1, 66.6 46.26 (2.09) 42.08-50.44 488.11 22.10 <.001 Group 1, 66.6 5.22 (2.86) −0.49 to 10.94 3.33 1.82 .07 Linear 1, 58.0 −2.29 (4.06) −10.42 to 5.84 0.32 −0.56 .57 Quadratic 1, 52.1 1.11 (2.00) −2.90 to 5.14 0.31 0.56 .58 Linear × Group 1, 59.2 −21.87 (5.92) −33.73 to −10.02 13.63 −3.70 <.001 Quadratic × Group 1, 50.8 7.47 (2.90) 1.63-13.30 6.61 2.57 .01 Difference POST −9.16 (3.50) −16.09 to −2.23 6.86 .01 d POST 0.89 0.35-1.43 Difference 12mFU −8.07 (3.67) −15.33 to −0.8 4.84 .03 d 12mFU 0.78 0.25-1.32 BDI-II c Intercept 1, 63.9 19.33 (1.45) 16.43-22.22 177.50 13.32 <.001 Group 1, 64.2 1.62 (1.99] −2.36 to 5.59 0.66 0.81 .42 Linear 1, 37.5 0.41 (0.92) −1.47 to 2.28 0.19 0.44 .66 Quadratic - - - Linear × Group 1, 36.1 −5.58 (1.31) −8.23 to −2.92 18.12 −4.26 <.001 Quadratic × Group - - - Difference POST −3.91 (1.89) −7.68 to −0.13 4.29 .04 d POST 0.52 −0.003 to 1.04 Difference 12mFU −9.41 (2.49) −14.34 to −4.48 14.31 <.001 d 12mFU 1.25 0.69-1.82 BAI d Intercept 1, 57.8 11.48 (1.52) 8.44-14.51 57.36 7.57 <.001 Group 1, 57.9 5.11 (2.08) 0.95-9.26 6.06 2.5 .02 Linear 1, 33.0 1.84 (0.83) 0.15-3.52 4.93 2.2 .03 Quadratic - - - Linear × Group 1, 32.2 −6.06 (1.17) −8.44 to −3.67 26.80 −5.2 <.001 Quadratic × Group - - - Difference POST 0.94 (1.98) −4.91 to 3.03 0.23 .23 d POST 0.12 −0.39 to 0.64 Difference 12mFU −6.99(2.50) −11.94 to −2.04 7.85 .006 d 12mFU 0.92 0.38-1.46 a SE: standard error. b PCL-C: PTSD-Checklist-Civilian version. c BDI-II: Beck Depression Inventory-II. d BAI: Beck Anxiety Inventory. Figure 2Estimated outcomes from mixed models with continuous variables. BODY.RESULTS.OUTCOMES: Table 3 and Figure 2 present the results from the mixed effects models and effect sizes for the continuous outcomes. BODY.RESULTS.OUTCOMES.PRIMARY OUTCOME: For PCL-C, a model with a quadratic term, indicating a nonlinear development over time, provided best fit to the data (see Multimedia Appendix 1). The intervention group exhibited a significant decline that abated over time. The control group showed no change. The model difference at postassessment was 9.16 points on the PCL-C in favor of the intervention group, representing a large effects size. At follow-up, the model difference was 8.07 points on the PCL-C in favor of the intervention group, representing a medium to large effect size. BODY.RESULTS.OUTCOMES.SECONDARY OUTCOMES: For BDI-II, a linear model provided best fit to the data (see Multimedia Appendix 2). The intervention group exhibited a significant decline over time. The control group showed no change. The model difference at postassessment was 3.91 points on the BDI-II in favor of the intervention group, representing a medium effect size. The model difference at follow-up was 9.41 points on the BDI-II, representing a large effect size. For BAI, a linear model provided best fit to the data (see Multimedia Appendix 3). At preassessment, the intervention group reported a significantly higher level of symptoms; the model difference was 5.11 points on the BAI. The intervention group exhibited a significant decline over time, whereas the control group exhibited a significant increase in symptoms over time. At postassessment, there was no difference between the groups. However, at follow-up there was a difference of 6.99 points on the BAI in favor of the intervention group, representing a large effect size Results for the economic variables are presented in Multimedia Appendix 4. Significantly more parents in the intervention group had seen a social worker at preassessment. No other differences between the groups were evident. BODY.DISCUSSION: To the best of our knowledge, this is the first study to report on the long-term outcomes of guided self-help via the Internet for parents of children on cancer treatment. The results indicate that the intervention was effective in terms of reductions in the primary outcome PTSS and that these improvements were maintained at the 12-month follow-up. Significant reductions for the intervention group compared with the control group were also evident for the secondary outcomes of depression and anxiety, and between-group effect sizes were even larger at the 12-month follow-up. However, there was no support for the intervention being effective in reducing health care consumption or sick leave. The findings that psychological distress can be reduced among parents of children with cancer are in line with previous investigations on problem-solving training administered in face-to-face format for mothers [11,12]. In the trial of problem-solving training compared with an inactive control condition [11], similar standardized mean differences between groups, as outlined in this study as metrics of effect-sizes, were not presented, which makes it hard to compare the treatment effects of these two trials. However, in the problem solving trial, significant intervention effects were observed for PTSS and depression at the end of the intervention (3 months post randomization), and these differences were maintained at the follow-up, 6 months post randomization. The results presented herein extend these findings and suggest that significant intervention effects for PTSS and depression observed post intervention can be maintained (PTSS) and seemingly improved (depression) at 12-month follow-up. In addition to mode of administration, another important difference between the two trials is that the current trial included participants reporting an increased level of PTSS, whereas the problem-solving trial included mothers irrespective of self-reported level of distress. Arguably, this results in different populations, which suggest that comparisons between the trials should be made with caution. We did not find support for treatment efficacy in terms of reduced health care consumption or sick leave. Importantly, the small sample size and substantial attrition made it difficult to use estimation techniques that are in line with the ITT principle (eg, general estimating equations), and we were forced to base the analysis on completers. Furthermore, the frequencies of some of the indicators for health care consumption and sick leave were small and with little variation. It is also important to note that previous research regarding economic strains on families caring for a child with cancer has mainly concerned working hours and expenses [5,6]. Such factors may well impact on parents level of distress as economic burden may be an additional stressor for the parents. However, psychological distress may not impact on working hours and expenses. In this study, we strived to assess economic factors that may be influenced by the parents' level of distress, such as health care consumption and sick leave and to investigate whether reductions in psychological distress would be associated with reductions in health-related expenses or sick leave. Future research should aim to disentangle whether psychological distress contributes to strains on household economy among parents of children on cancer treatment. Such efforts should be able to better prepare research that aims to alleviate the economic burden imposed on parents on children with cancer, be it via psychosocial interventions or targeted policies. It may also be the case that we used a less than optimal instrument for assessing health-related consumption and sick leave. Future research is needed to develop reliable and valid instruments for assessing these factors in the current population, and such research could preferably start with qualitative methods in order to explore the phenomena of health-related costs in this population. For the clinical outcomes, including the primary outcome PTSS, we used ITT-analyses with maximum likelihood estimation under the assumption of data missing at random, and under such assumption, this procedure produces less biased estimates compared with, for example, last-observation carried forward [31]. The results from these analyses indicate that the intervention produces substantial long-term improvements in PTSS, depression, and anxiety. However, using an inactive control group makes it impossible to draw conclusions regarding the specificity of the intervention. Future trials should include active control conditions that allow for the control of the nonspecific factors (eg, attention, information, and social support) that psychological interventions may be associated with. Furthermore, the sample was small, and attrition was substantial, which may limit the validity of the ITT-analyses and may hamper both the reliability and generalizability of the findings. In addition, the study is limited by the fact that reasons for not participating in the trial were not documented due to ethical reasons, which further limits the generalizability. As the study was small and underpowered, the risk for inflated effect sizes and type-I errors is also increased [34], which should be kept in mind when interpreting the findings. It is our impression that the intervention might have been too reliant on text and perhaps too time-consuming for parents in this difficult situation and might have deterred many from participating and also affected attrition. Potential adjustments include shortening the intervention and relying less on text in favor of other ways of communicating on the Web. In addition, fathers were less likely to participate at the 12-month follow-up than mothers, which may further limit the generalizability. Unfortunately, the small sample size made it difficult to further explore the underlying mechanism of this difference in attrition. Historically, fathers have been less involved in pediatric psychology research [35], and future research with parents of children with cancer should take steps to ensure involvement from parents of both sexes [36]. Finally, this study relied on self-reported outcomes, and participants were not blind to their study condition, which should be kept in mind when interpreting findings. This is a problem for all research on psychological interventions using inactive control conditions, and the fact that participants were aware of their study condition might have affected their expectancies for improvement differently. In this study, no such expectancies were assessed so we cannot estimate or control for this factor in the analyses. Nonspecific factors such as expectancy for improvement might contribute to improvements in all kinds of psychological interventions. An alternative design with an active comparison condition receiving some kind of support, and also assessing treatment credibility and expectancies for improvement in both conditions, would had enabled us to better control for these factors. To the best of our knowledge, this paper presents the first trial of an intervention administered via the Internet aiming to reduce psychological distress among parents of children on cancer treatment. ITT-analyses indicate substantial improvements in PTSS, depression, and anxiety that are maintained or strengthened at 12-month follow-up. However, we found no evidence for effects on health care consumption or sick leave. As such, using the Internet to provide psychological interventions may be an effective mode of delivery for parents reporting an increased level of PTSS and who consider Internet-based interventions as a viable option. Future research should corroborate these findings and also develop and evaluate interventions and policies that may help ameliorate the economic burden that parents may face during their child ́s treatment for cancer.

TITLE: Evaluation of Cyavanaprāśa on Health and Immunity related Parameters in Healthy Children: A Two Arm, Randomized, Open Labeled, Prospective, Multicenter, Clinical Study ABSTRACT.CONTEXT:: Cyavanaprāśa (CP) is an Ayurvedic immune booster formulation that confers vigor and vitality while delaying the ageing process. Benefits of CP have been studied widely in adult population. ABSTRACT.OBJECTIVES:: Current study assessed beneficial effects of CP on health and immunity related parameters in healthy children. ABSTRACT.METHODS:: This study was a 6 month long two armed, randomized, open labeled, prospective clinical study. School going healthy children between ages of 5-12 years were randomized to receive orally daily either CP (approx. 6 g) followed by a cup of milk (100 – 200 ml) or cup of milk only twice a day while continuing with their normal/routine diet. Results were analyzed based on number of episodes, severity, duration of illness (infections and allergies) and number of absent days due to illness during the study duration and changes in levels of energy, physical fitness, strength, stamina and quality of life in children which were recorded in subject diary by their parents/Legally Acceptable Representative (LAR). ABSTRACT.RESULTS:: 702 participants were randomized, out of which 627 completed the study (CP n = 313; Control n = 314). Results of immunity (episodes of infections or allergy related conditions) showed more than 2 times protection from immunity related illness in CP Group as compared to the control. CP also showed better percentage improvement in energy levels, physical fitness, strength, stamina and quality of life assessed through KIDSCREEN QOL-27 questionnaires in children. ABSTRACT.CONCLUSION:: Regular consumption of CP for a period of six months could significantly improve immunity, energy levels, physical fitness, strength, stamina and quality of life in school going healthy children. ABSTRACT.STUDY REGISTRATION:: Clinical Trail Registry of India vide CTRI/2015/02/005574, Dated 24 February 2015. BODY.INTRODUCTION.BACKGROUND: Children suffer from infections more than adults. On an average, a child suffers from four to eight respiratory infections per year.[1] This number varies depending on the presence or absence of risk factors that predispose to an increase in infective or allergic agents exposure; such as day-care attendance, school-aged siblings, and second-hand smoke etc.[23] Children with atopic disease seem more likely to develop recurrent and persistent upper respiratory infections probably due to enhanced adherence of micro-organisms to inflamed respiratory epithelium.[4] It is estimated that consumers purchase about 95 million packages of OTC medication for cough and cold for use in such situations in children each year.[5] Cyavanaprāśa (CP) is an Ayurvedic health product that helps in boosting immunity. CP has been found to be effective as an immunity booster, vitalizer and a preventer of day to day infections and allergies such as common cold and cough etc.[6] In recent times, CP has gained immense popularity all over the world as a time tested immunity booster. CP can be consumed in all seasons as it contains weather friendly ingredients which nullify unpleasant effects due to extreme environmental and climatic conditions.[7] Beneficial effects of CP have been observed in nasal allergies and viral infections and seasonal influences.[789] There exists enough evidence suggesting its multifaceted biological activity in favor of positive health. Till now CP has been studied in diverse population and there is scarcity of documented data regarding its use in children, specially, school going children.[9] BODY.INTRODUCTION.OBJECTIVE: The current study was planned to study comprehensively the beneficial effects of CP in school going children for various immunity related health parameters. BODY.METHODS.STUDY DESIGN: Current study was a 6 month long two arm, randomized, open labeled, prospective, clinical study conducted between Mar 2015 to Feb 2016 at two school sites in Maharashtra, India viz. Sane Guruji Vidya Mandir, Hadapsar and Shishuvihar pre-primary and primary school, Akrudi that were attached to Sumatibhai Shah Ayurvedic Hospital, Hadapsar and PDEA's College of Ayurveda and Research Center, Akrudi, respectively. The study was conducted in compliance with applicable ethical guidelines. Ethics committee approvals were obtained before study initiation. The Study was registered with the Clinical Trail Registry of India vide CTRI/2015/02/005574, Dated 24 Feb 2015. The CONSORT statement guidelines have been followed in reporting the outcomes of the study.[10] BODY.METHODS.STUDY PARTICIPANTS: School going children of either sex were screened for eligibility (Day 3) and were recruited if they fulfilled the inclusion and exclusion criteria. Written informed consent was taken from parents/Legally Acceptable Representatives (LAR) along with participant assent where applicable. BODY.METHODS.STUDY PARTICIPANTS.INCLUSION CRITERIA: Healthy children of either sex aged between 5-12 years, having no history of allergic conditions to CP like products or any ingredient of CP and who were not averse to consuming CP for the study duration were included. BODY.METHODS.STUDY PARTICIPANTS.EXCLUSION CRITERIA: (i) Children with presence of any clinically significant medical or surgical condition requiring long-term treatment; (ii) subjects participating/having participated in another clinical study within 3 months before recruitment; (iii) evidence of significant uncontrolled concomitant disease which in the investigator's opinion would preclude patient participation and (iv) immunologically compromised children were excluded. BODY.METHODS.STUDY INTERVENTIONS: Cyavanaprāśa is a classical Ayurvedic formulation comprising ingredients such as Pāṭalā (Stereospermum suaveolens, St. Bk.), Agnimantha (Clerodendrum phlomidis, St. Bk.), Gambhārī (Gmelina arborea, St. Bk.), Bilva (Aegle marmelos, St. Bk.), Syonāka (Oroxylum indicum, St. Bk.), Gokṣura (Tribulus terrestris, Fr.), Śālaparṇī (Desmodium gangeticum, Pl.), Prśṇiparṇī (Uraria picta, Pl.), Bṛhatī (Solanum indicum, Pl.), Kaṇṭakārī (Solanum surattense, Pl.), Pippalī (Piper longum, Fr.), Karkaṭaśṛṅgī (Pistacia integerrima, Gl.), Drākṣā (Vitis vinifera, Fr.), Guḍūcī (Tinospora cordifolia, St.), Harītakī (Terminalia chebula, P.), Balā (Sida cordifolia, Rt.), Tāmalakī (Phyllanthus niruri, Pl.), Vasā (Adhatoda vasica, Lf.), Jīvantī (Leptadenia reticulata, Pl.), Shaṭī (Heydichum spicatum, Rz.), Mustā (Cyperus rotundus, Rz.), Puṣkara (Inula racemosa, Rt.), Kākanāsikā (Martynia annua, Ft.), Mudgaparṇī (Phaseolus trilobus, Pl.), Māṣaparṇī (Teramnus labialis, Pl.), Punarnavā (Boerhavia diffusa, Rt.), Utpala (Nymphaea stellata, Fl.), Sūkṣmaila (Elettaria cardamomum, Sd., Ft. Tvak (Cinnamomum zeylanicum, Bk.), Candana Śveta (Santalum album, Ht. wd.), Vārāhī (Dioscorea bulbifera, Rt. Tr.), Śatāvarī (Asparagus racemosus, Rt.), Aśvagandhā (Withania somnifera, Rt.), Vidārī (Pueraria tuberosa, Rt. Tr.), Āmalakī (Emblica officinalis, Fr.), Ghṛta (Clarified butter), Tila taila (Sesamum indicum, oil), Śarkara (Crystal sugar), Madhu (Honey), Pippalī (Piper longum, Fr.), Vamśa (Bambusa bambos, S.C.), Tvakpatra (Cinnamomum tamala, lf.), Nāgakesara (Mesua ferrea, Stmn.), Abhraka Bhasma (Calcined mica), Muktā Piṣṭi (Anhydrous paste of pearl), Akarakarabha (Anacyclus pyrethrum, Rt.), Lavaṅga (Syzygium aromaticum, Fl.Bd.), Kuṅkuma (Crocus sativus, sty./stg.). In the current study, Cyavanaprāśa (Mfd: Dabur India Ltd.) manufactured following traditional methods[11] was used. The proximate analysis of CP showed Moisture content- 18.0—23.0% w/w, Total Fat- 1.5—2.5% w/w, Protein- Not detected, Dietary Fibre- 2.0—3.0% w/w, Total Ash- 3.0—5.0% w/w, Total Carbohydrate- 70.0—80.0% w/w. BODY.METHODS.STUDY PROCEDURE: At screening/baseline visit, a parent- teacher meeting was conducted to brief parents/LAR about study requirements and their written informed consent was obtained after one to one session with them. Assent for participation in the study was also obtained from participant, if applicable. Participants' physical and systemic examinations were done and vitals were recorded. Parents/LAR were handed over a subject diary and were trained to record episodes of illness (number of episodes, severity and duration of infection/allergies for assessment of immunity) and the KIDSCREEN QOL-27 Health Questionnaire in it. Recruited children were assigned to either CP group or Milk Group as per computer generated randomization list in 1:1 ratio. Children in CP Group were provided 500 gm jar of CP and parents/LAR were asked to give their child half a teaspoon (approx. 6 gm) of CP orally two times followed by a cup of milk daily. Parents in Milk group were asked to give their child a cup of milk only twice daily. The cup of milk was considered to be between 100-200 ml. Both the interventions were prescribed for a period for 6 months; during which, participants in both the groups were advised to continue with their normal/routine diet. A record of the children who were absent during the treatment period was observed and the reason for the same was recorded. BODY.METHODS.FOLLOW UP AND END OF STUDY ASSESSMENT: Participants were followed up for 6 months from baseline on Day 30, 60, 90, 120, 150 and 180 (±7 d). Last participant follow up was observed in the month of Feb 2016. At each follow-up visit, filled in questionnaires/scales were collected, study drug compliance was checked and CP supply was replenished as applicable. If 80% study medication was consumed over 80% time, the participant was considered compliant. Parents/LAR were enquired for any adverse event (s). At the end of the study, Parents/LAR were asked to stop giving CP and take the advice of investigator for further treatment, if any. BODY.METHODS.OUTCOMES: The primary outcome of the study was the assessment of immunity through episodes, severity and duration of immunity related illness (infections or allergies) in both study groups based on the subject diary. The secondary outcomes of the study were assessment of immunity through number of children having episode of immunity related illness; number of absent days and number of children absent from school due infection or allergy; children's Quality of life was evaluated using KIDSCREEN QOL-27 Health Questionnaires for Children. Their energy levels, physical fitness, strength and stamina; physician's global evaluation for overall improvement on CGI-I Scale; incidence of adverse events, serious adverse events and vitals during the study period; overall safety and tolerability by the physician and participant/parent was evaluated at the end of the study. Prakṛti evaluation was also conducted at the screening/baseline visit. Levels of Energy, physical strength, stamina and, the physical fitness were measured on a 7-point scale ranging from +3 to -3 [-3 (very dissatisfied), -2 (somewhat dissatisfied), -1 (a little dissatisfied), 0 (neutral), +1 (a little satisfied), +2 (somewhat satisfied), and +3 (very satisfied)]. KIDSCREEN QOL-27 Health Questionnaires were scored based on the scale ranging from 1 to 5 (1 - Excellent, 2 - Very Good, 3 - Good, 4 -Fair, 5- Poor; 1 - Not at all, 2 - Slightly, 3 - Moderately, 4 - Very, 5 - Extremely) depending on the response of the questionnaire). BODY.METHODS.STATISTICAL ANALYSIS: The primary population for this study was Per-Protocol population. With more than 300 participants in each group, the sample size was considered sufficient for analysis. The primary efficacy endpoints were analyzed using two proportion test. A 95% confidence interval was constructed for the proportion. All secondary outcomes were analyzed by applying appropriate statistical methods such as proportion test, t-test etc. BODY.RESULTS AND OBSERVATIONS: A total of 703 school going children were screened in the study of which one was a screen failure and 702 were randomized. Of the 702 participants, there were 75 dropouts while 627 participants completed the study. Drop out reasons included (i) withdrawal of consent, (ii) Investigational Product (IP) taste dislike, (iii) lost to follow-up, (iv) change of school due to shifting of home to a different place or job transfer of parent to a far away place from current location etc., None of the drop-outs was due to adverse event. Out of the participants who completed the study, 313 participants were in CP Group and 314 were in Control Group. There were a total of 325 males (51.83%) and 302 females (48.16%). Of the 325 males, 161 (51.43%) were in CP group while 164 (52.22%) were in the control group while out of the 302 females, 152 (48.56%) were in the CP group while 150 (47.77%) were in the control group. Only those participants who completed the total study duration were included in the analysis. The average age of participants in the CP group was 7.31 ± 1.75 while in the control group it was 7.36 ± 1.80. The participant flow chart for the study is shown in Figure 1. Figure 1Patient enrollment flow chart: CONSORT 2010 flow diagram The Prakṛti wise distribution of participants is shown in Table 1. It was observed that a majority of participants in both the groups were in the category of pitta-kapha, pitta-vāta and kapha-pitta prakṛti. There were 281 participants (89.77%) belonging to these phenotypes in CP group while in the control group; there were 284 participants (90.44%) of these phenotypes. Table 1 Prakṛti wise distribution of participants in the two groups BODY.RESULTS AND OBSERVATIONS.EFFICACY PARAMETERS: Primary and secondary outcomes of current study were observed in term of episodes of infection or allergy which were further divided into various categories [Table 2]. Table 2 Infection/allergy related disorders observed in the study in the two groups BODY.RESULTS AND OBSERVATIONS.EFFICACY PARAMETERS.ASSESSMENT OF IMMUNITY RELATED PARAMETERS: Assessment of episodes of infection or allergy A total of 1255 episodes related to infection or allergy were recorded in the study. Of these, 404 episodes (32.19%) were in CP group while 851 (67.80%) were in control group, which was assessed to be statistically significant (P < 0.001) and the proportionate difference was more than 2 times (2.10) when compared between the groups [Figure 2]. Figure 2Number of episodes of illness related to infection/allergy Evaluation of total duration of and severity of illnesses Evaluation of total duration of illnesses related to infection and allergy showed that of the total duration of 3983 days of illness, there were 1369 (34.37%) illness days in CP group whereas it was 2614 (65.62%) days in the control group. This difference was observed statistically significant (P < 0.01) and the proportionate difference was about 2 times (1.91) [Figure 3]. A proportionate difference of 1.25 to 2.37 fold were observed between CP and control group while assessing episodes and duration of illness related to infection or allergy in terms of severity [Table 3]. Figure 3Duration of illness related to infection/allergy Table 3 Assessment of episodes and duration of illness related to infection/allergy based on severity Assessment of number of participants having episodes of infection/allergy A total of 540 participants had episodes of infection/allergy related to immunity. Of these, 254 participants (47.03%) participants were in CP group while 286 (52.96%) were in the control group. The difference observed was statistically not significant (P > 0.05). 87 participants did not have any episode related to infection/allergy. Of these, 60 participants (68.96%) were in CP group while 27 (31.03%) participants were control group. The difference was statistically significant (P < 0.01). Absenteeism due to infection or allergy Numbers of absent days: There were a total of 702 absent days in 627 participants. Of these, 215 days (30.62%) were in CP group while 487 (69.37%) were in the control group. This difference was observed to be statistically significant (P < 0.001) [Figure 4]. There were a total of 281 participants who were absent due to infection/allergy/immunity related illnesses. Of these, 93 participants (33.09%) were in CP group while 188 (66.90%) were in the control group. This difference was found to be statistically significant (P < 0.001) [Figure 5]. Figure 4Assessment of number of absent days due to infection or allergy Figure 5Number of participants absent due to infection/allergy related illnesses BODY.RESULTS AND OBSERVATIONS.ASSESSMENT OF ENERGY, PHYSICAL FITNESS, STRENGTH AND STAMINA: Though no significant difference was observed between the groups at 3 and 6 months follow up, CP group showed a better improvement in energy levels, physical fitness, strength and stamina as compared to control group [Table 4]. Table 4 Assessment of energy, physical fitness, strength and stamina In CP group, there were 12 participants who were dissatisfied (Very dissatisfied, somewhat dissatisfied, A little dissatisfied) with their energy level at the baseline visit, which was reduced to two participants at the end of three months. At the end of six months, there was no participant who was dissatisfied with energy levels in CP Group. In the control group, however, there was one participant left dissatisfied out of 15 at baseline at the end of six months. 15 participants were dissatisfied with their physical fitness at the baseline visit in CP group; this was reduced to one participant at the end of three and six months. However, in control group one participant out of 16 at baseline was dissatisfied with his physical fitness participants at the end of 6 months. In CP group, 16 participants were dissatisfied with their strength at the baseline visit, which was reduced to one participant at the end of 3 months and none at the end of 6 months. In control group, however, one participant was left in dissatisfied out of 16 participants at baseline at the end of 6 months in control group. There were 14 participants who were dissatisfied with their stamina at the baseline visit in CP group, which was reduced to one participant at the end of 3 months and none at the end of 6 months. In control group, there was no dissatisfied participant out of 14 at baseline at the end of 6 months [Table 5]. Table 5 Assessment of number of participants showing change in energy level, physical fitness, strength and stamina BODY.RESULTS AND OBSERVATIONS.ASSESSMENT OF QUALITY OF LIFE THROUGH KIDSCREEN-27 QUESTIONNAIRE: Assessment of Quality of life through KIDSCREEN-27 Questionnaire over a period of 6 months showed an improvement in the domain score of "Physical Activity and Health" and "General Mood and Child's Feeling" in both the groups at the end of 6 months of the study [Table 6]. As per one previously conducted survey on children, KIDSCREEN QOL-27 health questionnaire has been found to be a valid tool for accessing child's quality of life.[12] Table 6 Assessment of change in quality of life through KIDSCREEN-27 questionnaire BODY.RESULTS AND OBSERVATIONS.ASSESSMENT OF PARTICIPANTS ON CGI-I SCALE: Assessment of participants on CGI-I scale showed that a higher percentage of participants (66.12%) showed improvement on this scale in CP Group as compared to control group (51.43%) [Table 7]. Table 7 Physician's global evaluation for overall improvement on CGI-I scale BODY.RESULTS AND OBSERVATIONS.ASSESSMENT OF SAFETY.ASSESSMENT OF OVERALL SAFETY AND TOLERABILITY AS PER PHYSICIAN: Assessment of overall safety by the physician showed that of the total 313 participants who were in the CP group, 92.45% showed excellent safety while the remaining 7.55% participants showed good or fair overall safety [Table 8]. Table 8 Assessment of overall safety and tolerability of cyavanaprāśa as per physician BODY.RESULTS AND OBSERVATIONS.ASSESSMENT OF OVERALL SAFETY THROUGH ADVERSE EVENTS MONITORING: Adverse events which were not related to infection or allergy were recorded separately as AE in both the groups. A total of 248 participants were reported to have AE of which 133 were in CP group and 115 in the Control group. A total of 599 AE were recorded in the study, 316 in CP group and 283 in Control Group. These AE were, however, not found to be related to the study drugs i.e. there were no Adverse Drug Reaction in any participant. Fourteen events were assessed and reported as SAE, of which 6 were in CP group and 8 were from Control Group. None of the SAE's was related to the study drug or procedure. Assessment of vitals such as pulse, respiration and body temperature did not show any significant difference either within or between the groups. The study drug was well tolerated by the participant. BODY.DISCUSSION: In recent times, CP has gained immense popularity all over the world as a time-tested immunity booster. Various pre-clinical[1314151617] and clinical studies have been conducted to evaluate its safety and efficacy. Efficacy of CP in term of anti-allergic property, activity of Natural Killer (NK) cells and phagocytic activity, level of Tumor Necrosis Factor- alpha (TNF-a), IL- 1-b and MIP-1- a secreted by Dendritic Cells (DC,[14] improvement of quality of life in patient with recurrent cough and cold and health benefits under seasonal variations has been established.[89181920] The benefits of CP have been validated in adults. In the current study, recruited children were randomized into two groups in which male and females were almost equally divided. Demography parameters analysis showed that age and gender were equally represented in both groups. The Prakṛti classification of the majority of participants enrolled in the study were Pitta-Kapha, Pitta-Vāta and Kapha-Pitta which were almost equally distributed in CP and control group. In the current study, CP significantly improved the immunity of school going children. The study was conducted from the month of Mar 2015 to Feb 2016 which covered one summer, one rainy and one winter season. It showed that the children were protected from various illnesses related to infection or allergy under the different seasonal variations though out the year. These findings are also in consensus with earlier published literature which demonstrated that CP provides protection from various illnesses irrespective of seasonal influence.[181920] Out of a total of 1255 episodes related to immunity related infections or allergies, control group had 2.10 times more number of episodes in comparison to CP group which was statistically significant (P < 0.01). Out of the total duration 3983 days of all episodes, the duration of such illness days was 1.91 times lesser in the CP group as compared to control group and was found to be statistically significant (P < 0.01). 540 participants were found to be affected by various illnesses related to infection or allergy and 87 participants did not have any such episode. The number of children having no immunity related illnesses was 2.22 times higher in the CP group as compared to control group which was found to be statistically significant (P < 0.01). Out of the total absent days from school, 2.27 times higher number of days were observed in the control group in comparison to CP group and found to be statistically significant (P < 0.001) as well. Out of the total participants who were absent from school due to such illness, 2.02 times more were observed in the control group and was found to be statistically significant (P < 0.001). These above mentioned effects are most likely related to the immune boosting potential of CP. The mechanism of these benefits of CP can be explained with the help of previously published literature and studies conducted on CP. CP has demonstrated the in vitro immune boosting potential by various mechanisms. In the concentration range of 20 μg/ml – 500 μg/ml, CP was found to increase the activity of NK cells by 1.8 to 10.3 fold, levels of TNF-α, IL- 1-β and MIP-1- α secreted by DCs were found to be increased by 1.3 to 3.2 fold and phagocytic activity was found to be increased by 28.9% to 65.2%.[1314] In two separate in-vivo studies, the level of IgE and plasma histamine were observed to be almost equal to the vehicle when challenged with allergens in animals (mice and rats respectively) pretreated with CP.[21] These two studies are suggestive of suppression of allergic response in response to allergens implying protection against various infections. Studies on CP have indicated that it contains a number of phenolics which may account for its therapeutic activity.[22] CP also contains honey which works as 'a carrier of herbs', or Yogavāhī, thereby potentiates absorption of various herbs deep into the tissues and imparts youth, charm, vigour and longevity.[7] In the current study as well, the subject diaries were evaluated to assess the change in energy, physical fitness, strength and stamina. Under all of these parameters, fair amounts of improvement were observed at end of three months that continued until the end of the study in both groups. The improvement was higher in CP group in comparison to control. However, comparison within and between the groups was not statistically significant. Assessment of above mentioned parameters in terms of number of children revealed that in CP group all participants except one, moved from the category of dissatisfied to satisfied at the end of the study in comparison to control where 4 participants were still in category of dissatisfied at the end of study. Assessment of Quality of life through KIDSCREEN-27 Questionnaires showed improvement in the domain score of "Physical Activity and Health" and "General Mood and Child's Feeling" in both the groups at the end of 6 months of the study. The domain score of "Physical Activity and Health" in CP as well as in control group improved from baseline to the end of the study and was found to be significant. The improvement in this domain was however found to be higher in CP group however; there was no significant difference between the two groups. Similarly the "general mood and child's feeling" showed a better improvement in CP group as compared to control group over 6 months period. The domain score of 'School & learning' showed a significant improvement in both the groups from one month further to 3 months and 6 months. The other domains of 'Family and child's free time and Friends' did not show any significant change both within the group and between the two groups. Assessment of participants on CGI-I scale showed that a higher percentage of participants showed improvement on this scale in CP group as compared to control group. Assessment of overall safety by the physician showed that of the total 313 participants in CP group, 92.45% of the participants showed excellent safety while the remaining 7.55% participants showed good or fair overall safety. All reported AEs and SAEs were found to be not related to the study drugs. Assessment of vitals did not show any significant finding. As per earlier published literature as well, CP was found to be safe in the recommended dosage for the defined period of time in toxicity studies. In all the toxicity studies i.e. acute dose oral toxicity, 28 days repeated dose oral toxicity studies and in the 90 days repeated dose oral toxicity studies, CP did not cause any signs of toxicity, body weight loss with all animals surviving throughout the study periods. In acute oral dose toxicity, the LD50 value of CP in both sexes of rats and mice was found to be greater than 2000 mg/kg of the body weight. The no observed adverse effect level (NOAEL) of CP was found to be 1000 mg/Kg body weight in both 28 day and 90 day toxicity studies.[13] The results of the above study on immunity related benefits of CP and safety parameters appear to be in line with the earlier studies and published data on the same. BODY.CONCLUSION: The present study concluded that regular consumption of CP for a period of three to six months helps to improve immunity, energy levels, physical fitness, strength, stamina and quality of life of children as assessed by various parameters. The primary parameter of immunity (episodes of infections or allergy related conditions) showed more than 2 times protection from illness in CP group as compared to the control group. The duration of illness related to infection or allergy was also observed to be significantly lesser in CP group as compared to control group. Other parameters of assessment of immunity such as number of absent days and number of absent participants in school due to these illnesses, also confirmed that CP showed more than twice the protection from immunity related illness as compared to the control group. There was a significant change in energy levels, physical fitness levels, strength and stamina in both the groups, however, CP showed better percentage improvement on these parameters as compared to the control group. Assessment of quality of life by KIDSCREEN-27 Questionnaire showed better improvement in CP group as compared to control group on domains of "Physical activity and Health", "General Mood and Child's Feelings" and "School and learning." Assessment of overall improvement by the physician (CGI-I) also showed that over a period of 6 months more number of participants improved in CP group as compared to control group. CP showed excellent to good safety in all the participants in the study. No Adverse events/Serious Adverse events reported was assessed to be related to study product or procedure. BODY.CONCLUSION.FINANCIAL SUPPORT AND SPONSORSHIP: The study was sponsored by Dabur Research and Development Centre, Dabur India Limited, Sahibabad (Ghaziabad) Uttar Pradesh, India. BODY.CONCLUSION.CONFLICTS OF INTEREST: Some of the Authors (Arun Gupta, Sunil Kumar and V. Sasibhushan) are currently employed with Dabur India Ltd.

TITLE: Efficacy and Safety of 5-Fluorouracil 0.5%/Salicylic Acid 10% in the Field-Directed Treatment of Actinic Keratosis: A Phase III, Randomized, Double-Blind, Vehicle-Controlled Trial ABSTRACT.INTRODUCTION: Due to the high prevalence of actinic keratosis (AK) and potential for lesions to become cancerous, clinical guidelines recommend that all are treated. The objective of this study was to evaluate the efficacy and safety of 5-fluorouracil (5-FU) 0.5%/salicylic acid 10% as field-directed treatment of AK lesions. ABSTRACT.METHODS: This multicenter, double-blind, vehicle-controlled study (NCT02289768) randomized adults, with a 25 cm2 area of skin on their face, bald scalp, or forehead covering 4–10 clinically confirmed AK lesions (grade I/II), 2:1 to treatment or vehicle applied topically once daily for 12 weeks. The primary endpoint was the proportion of patients with complete clinical clearance (CCC) of lesions in the treatment field 8 weeks after the end of treatment. Secondary endpoints included partial clearance (PC; ≥75% reduction) of lesions. Safety outcomes were assessed. ABSTRACT.RESULTS: Of 166 patients randomized, 111 received 5-FU 0.5%/salicylic acid 10% and 55 received vehicle. At 8 weeks after the end of treatment, CCC was significantly higher with 5-FU 0.5%/salicylic acid 10% than with vehicle [49.5% vs. 18.2%, respectively; odds ratio (OR) 3.9 (95% CI) 1.7, 8.7; P = 0.0006]. Significantly more patients achieved PC of lesions with treatment than with vehicle [69.5% vs. 34.6%, respectively; OR 4.9 (95% CI 2.3, 10.5); P < 0.0001]. Treatment-emergent adverse events, predominantly related to application- and administration-site reactions, were more common with 5-FU 0.5%/salicylic acid 10% than with vehicle (99.1% vs. 83.6%). ABSTRACT.CONCLUSIONS: Compared with vehicle, field-directed treatment of AK lesions with 5-FU 0.5%/salicylic acid 10% was effective in terms of CCC. Safety outcomes were consistent with the known and predictable safety profile. ABSTRACT.TRIAL REGISTRATION: NCT02289768. ABSTRACT.FUNDING: Almirall S.A. ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13555-016-0161-2) contains supplementary material, which is available to authorized users. BODY.INTRODUCTION: Actinic keratosis (AK) is a common skin condition characterized by dysplastic lesions of keratinocytes that have the potential to become malignant [1, 2]. Infiltrative transformation of AK grade III lesions to invasive squamous cell carcinoma (SCC) was believed to occur via a classical pathway involving sequential progression from grade I through to grade III AK. However, recent findings indicate that invasive SCC can expand directly from a grade I AK lesion [3]. Owing to the high prevalence of AK, the risk of lesions becoming cancerous, and the inability to predict which lesions will progress to SCC, this justifies the treatment of all AK lesions regardless of grade [4, 5]. Current options for the topical treatment of AK lesions include diclofenac, hyaluronic acid, 5-fluorouracil (5-FU), imiquimod, and ingenol mebutate [6–8]. Lesion- or field-directed therapy is indicated subject to the specific characteristics of the lesions to be treated (e.g., their number, localization, extent, and clinical course) and patient features (e.g., age, comorbidities, and other risk factors) [9, 10]. Historically, lesion-directed treatments have been the most common approach for treating a single lesion, whereas field-directed therapies, which aim to treat areas with multiple AK lesions of varying degrees of severity, including sub-clinical (non-visible) lesions [9], are now preferred if lesion and patient characteristics permit [11]. 5-FU 0.5%/salicylic acid 10% (Actikerall®, Almirall S.A.) is indicated for the topical treatment of slightly palpable and/or moderately thick hyperkeratotic AK lesions (grade I/II according to Olsen et al. 1991 [12]) in immunocompetent adults, with an option to simultaneously treat multiple AK lesions, up to a maximum of 10 lesions in a total skin area of 25 cm2 [13]. However, only a maximum rim of 0.5 cm of healthy skin surrounding the lesions can come into contact with 5-FU 0.5%/salicylic acid 10%. Here we report new efficacy and safety data from the first randomized controlled trial investigating the efficacy and safety of 5-FU 0.5%/salicylic acid 10% when applied as field-directed treatment to a contiguous area of 25 cm2 in a field cancerization area on the face, bald scalp, or forehead of patients with 4–10 clinically confirmed AK lesions (grade I and II). BODY.METHODS.STUDY DESIGN AND ETHICAL CONDUCT: This phase III, multicenter, randomized, double-blind, vehicle-controlled study (ClinicalTrials.gov identifier: NCT02289768) was conducted at 14 sites in Germany and the UK. There were five treatment visits and a follow-up visit 8 weeks after the final treatment application, regardless of whether a patient had completed 12 weeks of treatment or prematurely discontinued from treatment (Fig. 1). Findings from a sub-study that examined the effect of 5-FU 0.5%/salicylic acid 10% on sub-clinical AK lesions using reflectance confocal microscopy (RCM) in a group of 30 patients will be published separately.Fig. 1Study design. *Once-daily topical administration. 5-FU 5-fluorouracil, V visit The study was conducted in accordance with the recommendations of the Helsinki Declaration of 1964, as revised in 2008, complied with International Conference on Harmonisation Good Clinical Practice guidelines and local regulations and was approved by an independent ethics committee. Informed consent was obtained from all patients in writing prior to inclusion in the study. BODY.METHODS.PATIENTS: Male and female (non-pregnant, non-lactating in the last 3 months) patients were enrolled in the study if they were aged 18–85 years and had 4–10 clinically confirmed AK lesions (grade I/II [12]) within a field cancerization area of 25 cm2 located on the face, bald scalp, or forehead. Patients had Fitzpatrick skin type I–IV, were in good health and were free of physical and mental conditions that could interfere with the examination or evaluation of the potential treatment area. During the study, patients had to refrain from sunbathing and avoid intense ultraviolet-light exposure/solarium. They also had to avoid the use of moisturizers and topical treatments with anti-aging products, ointments containing vitamins A, C, and/or E, and gels and green-tea preparations in the treatment area. Patients also had to be physically able (or have a supportive person) to apply the study preparations correctly and to follow the study procedure and restrictions. Key exclusion criteria included patients who ≤3 months before screening had received treatment for AK within the treatment area or if they had dermatological diseases in the treatment area or surrounding area that could be exacerbated by study treatment or could interfere with the study assessments (e.g., psoriasis, eczema). Additionally, patients were excluded if they had received topical treatment with certain pharmacological and non-pharmacological products (e.g., retinoids, steroids, diclofenac or 5-FU preparations, curettage, photodynamic therapy, and chemical peel) in the treatment area 4–8 weeks (depending on the product) prior to randomization. Patients were also excluded if they had received systemic medication (varying between 4 and 12 weeks, depending on the medication), including: interferons; immunomodulators or immunosuppressive drugs; diclofenac or 5-FU preparations; and cytotoxic drugs. The use of phenytoin, methotrexate, or sulfonylurea was also not allowed. BODY.METHODS.TREATMENT: At visit 2, patients were randomized 2:1 in ascendant chronological order to receive topical 5-FU 0.5%/salicylic acid 10% or colour/consistency/appearance-matched vehicle, which was self-administered once daily for 12 weeks using a brush enclosed in the cap of the medication container. This was a double-blind study so neither the patient nor the research personnel knew the treatment assigned to each patient. Study medication was applied at the same time each day according to the instructions in the product patient leaflet, except that the area of application was a contiguous 25 cm2. The dose regimen could be decreased by the physician to three doses per week in the case of severe local skin reactions. Treated areas were left uncovered, and study medication was allowed to dry to enable a film to form over the area. Prior to daily re-application of the medication, the film coating was peeled off and the skin washed with water and a damp cloth. Patients were instructed to not apply medication to bleeding lesions. The location of the treated area was chosen according to the ability of the patient to conveniently apply study medication on a daily basis. A plastic template was used to mark and later identify the same 25 cm2 treated area. The first and final treatment applications were performed at the study center by study staff. BODY.METHODS.STUDY ASSESSMENTS AND ENDPOINTS: The primary endpoint was the proportion of patients with complete clinical clearance (CCC) of AK lesions in the treatment field at 8 weeks after the end of treatment. CCC of AK lesions at each treatment visit (i.e., after 2, 4, 6, and 12 weeks of treatment) was measured as a secondary endpoint. Additional secondary endpoints reporting the effect of treatment on lesions included: partial clearance (PC; defined as ≥75% reduction in clinically visible AK lesions) at each treatment visit and 8 weeks after the end of treatment; proportional change from baseline in the total number of AK lesions at each treatment visit and 8 weeks after the end of treatment; the number of lesions by AK grade severity at baseline and 8 weeks after the end of treatment; and proportional change from baseline in the total number of lesions at each treatment visit and 8 weeks after the end of treatment. A physician-reported outcome was global assessment of efficacy [Physician Global Assessment (PGA)] at each treatment visit and 8 weeks after the end of treatment. Patient-reported outcomes included patient satisfaction with treatment by recording individual domain scores (i.e., effectiveness, side effects, convenience, and overall satisfaction) of the Treatment Satisfaction Questionnaire for Medication (TSQM, version 1.4) at 8 weeks after the end of treatment and quality-of-life assessment by recording the change from baseline in total and individual domain scores (i.e., daily activities, leisure, personal relationships, symptoms and feelings, treatment, work, and school) of the Dermatology Life Quality Index (DLQI) after 12 weeks of treatment and at 8 weeks after the end of treatment. DLQI was calculated by summing the score of each question resulting in a maximum of 30 and a minimum of 0, and the higher the score, the more quality of life is impaired. Adverse events (AEs) were collected from the time of informed consent to the follow-up visit; this was always performed 8 weeks post last treatment, regardless of whether the patient had completed the 12-week treatment period or had prematurely discontinued from the study. The reporting of AEs was elicited by asking patients non-leading questions and by collecting information regarding the AEs spontaneously reported by the patients to study staff. BODY.METHODS.STATISTICAL ANALYSIS: It was determined that a total of 146 patients (97 receiving active treatment, 49 receiving vehicle) was required to complete the study to provide 80% power to detect as "significant" a difference of 25% between 5-FU 0.5%/salicylic acid 10% and vehicle in the proportion of patients with CCC, assuming a clearance rate in the active group of 50% and a clearance rate in the vehicle group of 25%. To allow for an estimated 10% dropout rate, approximately 162 patients (108 receiving treatment, 54 receiving vehicle) were to be randomized. The primary endpoint (CCC) comparison of 5-FU 0.5%/salicylic acid 10% versus vehicle was analyzed using the Cochran-Mantel-Haenszel test statistic adjusting for anatomical site (face/scalp) and baseline (number of AK lesions). The number and proportion of responders for each treatment group, odds ratio (OR), corresponding 95% confidence interval (CI), and the two-sided P value associated with the Cochran-Mantel-Haenszel test were calculated. The 95% CI for the proportion of patients with CCC was calculated using the exact binomial test. The secondary endpoint of the proportion of patients with CCC of AK lesions in the treatment field at each treatment visit was analyzed in the same way as the primary efficacy variable. Other secondary endpoints and safety data were analyzed using descriptive statistics. Missing efficacy data were handled using the last observation carried forward (LOCF). An analysis of covariance model with treatment arm and anatomical site as factors and baseline as covariate was used for the analysis of total and individual domain scores of the TSQI and DLQI. All secondary endpoints were analyzed using the intent-to-treat (ITT) population only. BODY.RESULTS.PATIENTS: This study was conducted between 17 October 2014 and 10 August 2015. Of the 175 patients screened, 166 were randomized. Of these, 111 patients received 5-FU 0.5%/salicylic acid 10% (108 patients in the safety and ITT populations) and 55 patients received vehicle (Fig. 2).Fig. 2Patient disposition. a Includes 3 patients who were counted only as randomized; b overall, 12 patients prematurely discontinued treatment but completed follow-up: 5-FU/SA, n = 9; vehicle, n = 3. c All patients who discontinued follow-up also discontinued treatment: 5-FU/SA, n = 6; vehicle, n = 2. 5-FU/SA 5-fluorouracil 0.5%/salicylic acid 10% Baseline demographics were similar between the groups, with slightly more female patients in the active treatment arm (14.8%) versus vehicle (7.3%; Table 1). At baseline, 56.6% of lesions were classified as grade I and 43.4% were classified as grade II; similar ratios of grade I and grade II AK lesions were present in each treatment arm.Table 1Patient baseline demographics and clinical characteristics (safety population)5-FU/SA (N = 108)Vehicle (N = 55)Total (N = 163)Age, years (mean, SD)71.8 (7.3)72.8 (6.9)72.2 (7.1)Gender, male (%)85.292.787.7Race, Caucasian (%)100100100BMI, kg/m2 (mean, SD)27.5 (3.8)27.2 (3.9)27.4 (3.8)Proportion of AK lesions by grade (Olsen et al. [12]) (%) Grade I56.157.656.6 Grade II43.942.443.4Skin type (Fitzpatrick) (%) Type I8.39.18.6 Type II78.783.680.4 Type III12.07.310.4 Type IV0.90.00.6Number of AK lesions (mean, SD)5.6 (1.4)5.6 (1.5)5.6 (1.4)Location of AK lesions (%) Bald scalp44.746.345.2 Face/forehead55.353.754.8 AK actinic keratosis, BMI body mass index, 5-FU/SA 5-fluorouracil 0.5%/salicylic acid 10%, SD standard deviation BODY.RESULTS.EFFICACY: The percentage of patients with CCC 8 weeks after the end of treatment (primary endpoint) was significantly higher in the 5-FU 0.5%/salicylic acid 10% arm compared with vehicle in both the ITT and per protocol (PP) populations [ITT LOCF: 49.5% vs. 18.2%; OR 3.9 (95% CI 1.7, 8.7) P = 0.0006 (Fig. 3a); PP LOCF: 55.1% vs. 19.6%; OR 5.1 (95% CI 2.1, 12.2) P = 0.0002]. During treatment, there were no significant differences between 5-FU 0.5%/salicylic acid 10% and vehicle (Fig. 3b); however, it should be noted that it can be difficult to assess lesion counts during treatment because of irritation at the site of administration.Fig. 3Proportion of patients with complete clinical clearancea of AK lesions in the treatment field a at 8 weeks after the end of treatment (intent-to-treat population) and b during treatment (after 2, 4, 6, and 12 weeks) and 8 weeks after the end of treatment (intent-to-treat population). *P = 0.0006. Analysis was performed using the Cochran-Mantel-Haenszel test, adjusting for anatomical site and baseline. The last observation carried forward was used for missing data; however, for 5-FU/SA, three patients had only baseline data available so it was not possible to replace the missing data. a Complete clinical clearance defined as all lesions cleared and lesion count of zero at each visit. 5-FU/SA 5-fluorouracil 0.5%/salicylic acid 10%, AK actinic keratosis Eight weeks after the end of treatment, the proportion of patients who achieved PC of AK lesions was significantly greater in the 5-FU 0.5%/salicylic acid 10% arm than in the vehicle arm [69.5% vs. 34.6%; OR 4.9 (95% CI 2.3, 10.5) P < 0.0001 (Fig. 4)]. The proportional reduction from baseline in the total number of AK lesions per patient was significantly greater with 5-FU 0.5%/salicylic acid 10% than with vehicle: 78.0% versus 46.9%, respectively; P < 0.0001 (Fig. 5). For both PC and reduction in lesion count, there were no significant differences between the treatment groups at each visit of the 12-week treatment period. At 8 weeks after the end of treatment, a higher proportion of AK lesions in the active treatment arm had transitioned from grade I/II to grade 0 compared with the vehicle arm (Fig. 6).Fig. 4Proportion of patients with partial clearancea of AK lesions in the treatment field during treatment (after 2, 4, 6, and 12 weeks) and 8 weeks after the end of treatment (intent-to-treat population). *P < 0.0001. a Partial clearance defined as ≤75% reduction in the number of clinically visible AK lesions. 5-FU/SA 5-fluorouracil 0.5%/salicylic acid 10%, AK actinic keratosis Fig. 5Proportional change from baseline in the total number of AK lesions recorded during treatment (after 2, 4, 6, and 12 weeks) and at 8 weeks after the end of treatment (intent-to-treat population). *P < 0.0001. 5-FU/SA 5-fluorouracil 0.5%/salicylic acid 10%, AK actinic keratosis Fig. 6Proportion of AK lesions by severity a(according to Olsen et al. [12]) at a baseline and b 8 weeks after the end of treatment (intent-to-treat population). a 5-FU/SA 5-fluorouracil 0.5%/salicylic acid 10%, AK actinic keratosis Assessment of treatment efficacy according to PGA found that for 5-FU 0.5%/salicylic acid 10%, those with a PGA score of "good" or "very good" increased from 45.2% at week 2 to 90.2% at follow-up (Fig. 7). In contrast, this increased from 61.1% at week 2 to no more than 75.5% at follow-up for vehicle (P < 0.0001).Fig. 7Global assessment of efficacy by physician at each treatment visit (after 2, 4, 6, and 12 weeks) and 8 weeks after the end of treatment (intent-to-treat population). *P < 0.0001 (5-FU/SA vs. vehicle). 5-FU/SA 5-fluorouracil 0.5%/salicylic acid 10% At 8 weeks after the end of treatment, 5-FU 0.5%/salicylic acid 10% was associated with significant improvements in overall treatment satisfaction and effectiveness domain mean scores in the TSQM compared with vehicle [69.2 vs. 56.1 (P = 0.0019); 70.8 vs. 59.2 (P = 0.0064), respectively]. No statistically significant differences were observed between the study arms for the TSQM convenience (70.7 and 71.2, respectively) and side effect (92.4 and 96.4, respectively) domain scores. The clinical impact of AK lesions on the DLQI individual domains at baseline was low, with symptoms and feelings being the most affected. As shown in Table 2, improvement in DLQI total score was statistically greater for vehicle versus 5-FU 0.5%/salicylic acid 10% during the treatment phase; this was attributed to local skin reactions associated with 5-FU 0.5%/salicylic acid 10%. However, the improvement in DLQI total score switched in favor of 5-FU 0.5%/salicylic acid 10% 8 weeks after the end of treatment, although this was not statistically significant (P = 0.0725; Table 2; Supplementary Fig. S1).Table 2LS mean change from baseline in DLQI questionnaire scores at week 12 and 8 weeks after end of treatment5-FU/SA (N = 108)Vehicle (N = 55) P valueDLQI total score Week 120.53−0.3270.0052 8 Weeks post-treatment−0.667−0.1330.0725Daily activities Week 120.117−0.0410.0564 8 Weeks post-treatment−0.0490.1670.0294Leisure Week 120.105−0.0610.109 8 Weeks post-treatment−0.036−0.0050.6595Personal relationships Week 120.008−0.0830.1108 8 Weeks post-treatment−0.0760.0050.0934Symptoms and feelings Week 120.194−0.2480.0084 8 Weeks post-treatment−0.488−0.3390.3209Treatment Week 120.0890.1660.2729 8 Weeks post-treatment0.001−0.0070.8854Work and school Week 120.001−0.020.4985 8 Weeks post-treatment−0.0320.070.0371Analysis was based on an ANCOVA model in the change from baseline in total score and individual domains of the DLQI questionnaire adjusted by the correspondent baseline as covariate and treatment group and anatomical site as factors. The DLQI is calculated by summing the score of each question, resulting in a maximum of 30 and a minimum of 0. The higher the score, the more quality of life is impaired. In the 5-FU/SA group, for all scores n = 91–92 at week 12 and n = 100–101 at 8 weeks post treatment; in the vehicle group n = 50–51 at week 12 and n = 53–54 at 8 weeks post-treatment 5-FU/SA 5-fluorouracil 0.5%/salicylic acid 10%, ANCOVA analysis of covariance, DLQI Dermatology Life Quality Index, LS least squares BODY.RESULTS.SAFETY: The incidence of treatment-emergent AEs (TEAEs) was slightly higher in the 5-FU 0.5%/salicylic acid 10% study arm compared with vehicle (99.1% vs. 83.6%, respectively) (Table 3). This was predominantly a consequence of application- and administration-site reactions, occurring in 99.1% and 76.4% of respective patients in the active treatment and vehicle arms; the majority of these were of mild or moderate intensity. Nine (5.4%) patients had 11 treatment-emergent serious AEs (TESAEs): in the 5-FU 0.5%/salicylic acid 10% arm, 6 (5.6%) patients had a total of 8 TESAEs, and, in the vehicle arm, 3 (5.5%) patients had a total of 3 TESAEs.Table 3Incidence of TEAEs according to preferred term (safety population)TEAE, n (%)a 5-FU/SA (N = 108)Vehicle (N = 55)Total (N = 163)Application site Erythema96 (88.9)29 (52.7)125 (76.7) Pain75 (69.4)23 (41.8)98 (60.1) Irritation64 (59.3)15 (27.3)79 (48.5) Inflammation60 (55.6)15 (27.3)75 (46.0) Scab63 (58.3)12 (21.8)75 (46.0) Erosion46 (42.6)6 (10.9)52 (31.9) Pruritus36 (33.3)16 (29.1)52 (31.9) Dermatitis34 (31.5)3 (5.5)37 (22.7) Bleeding26 (24.1)3 (5.5)29 (17.8) Edema17 (15.7)0 (0.0)17 (10.4) Ulcer3 (2.8)0 (0.0)3 (1.8)Application-site exfoliationb 6 (5.6)3 (5.5)9 (5.5)Nasopharyngitisc,d 12 (11.1)2 (3.6)14 (8.6)Nasopharyngitisc,e 4 (3.7)1 (1.8)5 (3.1)Headache4 (3.7)2 (3.6)6 (3.7)Includes predefined local skin reactions (application-site TEAEs) that were anticipated from the known safety profile of 5-FU/SA 5-FU/SA 5-fluorouracil 0.5%/salicylic acid 10%, TEAE treatment-emergent adverse event aAll events occurred ≤30 days after the final treatment application bLocal skin reaction cNasopharyngitis identified from different system organ class high-level term dUpper respiratory tract infection eUpper respiratory tract infection, not elsewhere classified Treatment was discontinued as a result of a TEAE in two (1.9%) patients in the 5-FU 0.5%/salicylic acid 10% study arm; no patients in the vehicle arm discontinued treatment because of TEAEs. One discontinuation was because of application-site bleeding, which was deemed to be related to treatment, and one discontinuation was because of bladder neoplasm, which was not deemed to be treatment related. There were no deaths during the study. BODY.DISCUSSION: Field-directed treatment of AK may offer the most effective means of eliminating both clinical and subclinical lesions, thereby preventing progression to invasive SCC [14]. Our study, the first randomized controlled trial of field-directed therapy with 5-FU/SA, showed that field-directed treatment of AK lesions with 5-FU 0.5%/salicylic acid 10% applied once daily via topical administration for 12 weeks was associated with significantly higher rates of CCC at 8 weeks after the end of treatment compared with vehicle. PC of AK lesions and the proportional reduction from baseline in total number of AK lesions followed similar trends. In addition, at 8 weeks after the end of treatment, the severity of AK lesions was reduced to a greater extent with 5-FU 0.5%/salicylic acid 10% than with vehicle. The efficacy of 5-FU 0.5%/salicylic acid 10% applied as lesion-directed therapy for a maximum of 12 weeks with 8-week follow-up was explored previously in a phase III study in which it was used for the treatment of patients with AK lesions of similar grade and location to those in the present study [15]. In the previous study, histological clearance at 8 weeks, the primary study objective, was achieved in 72.0% of patients, a significantly higher rate than that achieved following treatment with diclofenac 3% in hyaluronic acid (59.1%; P < 0.01) and vehicle (44.8%; P < 0.0001). Low-dose 5-FU 0.5%/salicylic acid 10% was also superior to diclofenac 3% in hyaluronic acid and vehicle in terms of CCC (55.4% vs. 32.0% and 15.1% for diclofenac 3% in hyaluronic acid and vehicle, respectively; P < 0.001 for both comparisons). In a 12-month follow-up study [16], the percentage of sustained clearance of AK lesions was higher for 5-FU 0.5%/salicylic acid 10% (85.8%) versus diclofenac 3% in hyaluronic acid (81.0%; P = 0.02) and vehicle (79.8%; P = 0.04). A recent review of topical treatments for AK lesions suggested that 5-FU 0.5%/salicylic acid 10% was associated with a higher incidence of CCC than imiquimod 2.5% and 3.75% and ingenol mebutate [17]. It should be noted that the 5-FU 0.5%/salicylic acid 10% treatment group also included patients with hyperkeratotic lesions, which are more severe and have a potentially higher rate of malignant transformation [18, 19]; these were not included in the imiquimod and ingenol mebutate studies [17]. Longer-term clinical trials need to be carried out to validate these findings. At the end of the active treatment period, lesion counts were similar between 5-FU 0.5%/salicylic acid 10% and vehicle arms, a finding that may be attributable to ongoing difficulties in lesion assessment caused by irritation at the site of administration of both 5-FU/SA and vehicle. However, it is also possible that vehicle itself may have had a mild therapeutic effect mediated via an unknown mechanism; DMSO has a known irritant effect [13] and may thus activate the skin's defense mechanisms [15]. Eight weeks following the end of treatment, 5-FU 0.5%/salicylic acid 10% was associated with a significant 78% decrease in the number of AK lesions. This is comparable to the corresponding decrease in lesion count of approximately 70% observed in a non-interventional study of 1051 patients [20]. It should be borne in mind that 48.6% (498/1025) of patients in the study by Szeimies and colleagues received 5-FU 0.5%/salicylic acid 10% treatment for less than 6 weeks, which may account for the slight difference in lesion count reduction. Given that treatment with 5-FU 0.5%/salicylic acid 10% was associated with a greater number of lesions transitioning from grade I/II to grade 0 than with vehicle, this suggests that active treatment affects the histopathology of the AK lesions. Two small (case series) studies using RCM recently determined that field-directed 5-FU 0.5%/salicylic acid 10% for up to 6 weeks of treatment was effective not only for investigating the transitioning of clinically visible lesions from a higher to lower grade, but also for clearing sub-clinical lesions after field-directed treatment [21, 22]. The results from the RCM sub-study in a group of 30 patients from the current study will be published separately. At 8 weeks after the end of treatment, PGA of treatment efficacy was rated as "very good" or "good" in 90.2% of patients receiving 5-FU 0.5%/salicylic acid 10%. This was similar to the 89% reported in the non-interventional study by Szeimies and colleagues [20]. Overall treatment satisfaction, as measured using the TSQM questionnaire, was greater among patients in the 5-FU 0.5%/salicylic acid 10% arm than in the vehicle arm (P = 0.0019), as was satisfaction in relation to treatment effectiveness (P = 0.0064). In the 12-month follow-up study by Stockfleth et al., it was reported that 93.2% of patients receiving 5-FU 0.5%/salicylic acid 10% assessed treatment efficacy as "very good" or "good" compared with 81.6% in the diclofenac 3% in hyaluronic acid group and 66.7% in the vehicle group [16]. Although improvement in DLQI total score was statistically higher for vehicle versus 5-FU 0.5%/salicylic acid 10% during the treatment phase, this switched in favor of 5-FU 0.5%/salicylic acid 10% 8 weeks after the end of treatment and was attributed to local skin reactions associated with 5-FU 0.5%/salicylic acid 10%. Safety data in our study were consistent with the known and predictable tolerability profile of 5-FU 0.5%/salicylic acid 10% [15, 16]. As with other topical treatments for AK [23, 24], 5-FU 0.5%/salicylic acid 10% caused administration-site reactions, such as erythema, inflammation, and scabbing, prior to demonstrating clear evidence of efficacy. Based on our clinical experience and with the vast majority of topical treatments for AK, local adverse effects are expected and proportionally correlate with treatment duration and efficacy. For some patients who withdrew from treatment, administration-site local skins reactions were mentioned but not cited as the main reason for drug withdrawal; six patients (5.6%) in the 5-FU 0.5%/salicylic acid 10% group and two patients (3.6%) in the vehicle group. The number of patients for whom the investigator considered that administration-site local skin reactions justified treatment discontinuation was low (5-FU 0.5%/salicylic acid 10% arm: n = 1; vehicle arm: n = 0). Conservatively taking these numbers into account, treatment discontinuation rates due to safety/tolerability issues were low in this study. One potential limitation of our study was the higher incidence of local skin reactions in the 5-FU 0.5%/salicylic acid 10% arm, which may have compromised the blinding of the study. However, the incidence of local skin reactions in the vehicle group, which are similar to those reported previously in a pivotal clinical trial using a lesion-directed approach [15] and may have been linked to the irritant effects of dimethyl sulfoxide that are part of the known safety profile of 5-FU 0.5%/salicylic acid 10% [13], minimized the unblinding risk. Also, during the 8-week period after the end of treatment, patients were expected not to use further treatments for AK lesions, which may have proven difficult for some patients. However, the specification that patients had grade I/II lesions, rather than more severe AK, may have helped avoid the situation where additional treatment was required. While it is clearly important to evaluate clinical endpoints, selecting endpoints that only included clinical evaluation of lesions could be regarded as a potential limitation of the study. Good correlation between routine histology and the results of RCM, a novel non-invasive imaging technique that allows the in vivo evaluation of skin at near-histological resolution, has been reported previously [25]. Results from the RCM sub-study in a group of 30 patients from the current study will be published separately. A strength of the present study was the inclusion of patients with hyperkeratotic lesions, which enables demonstration of the efficacy of 5-FU 0.5%/salicylic acid 10% against these more clinically severe lesions. In addition, the findings from this first randomized study with field-directed therapy with 5-FU/SA are robust, with consistency across all reported variables, both objectively measured and patient-reported. BODY.CONCLUSION: In this randomized, double-blind, vehicle-controlled study, once-daily topical treatment with field-directed 5-FU 0.5%/salicylic acid 10% applied over a 12-week period was an effective treatment for grade I and II AK lesions. Furthermore, treatment was well accepted by both physicians and patients in terms of overall treatment satisfaction and clinical outcomes. In addition, safety outcomes were consistent with the known and predictable safety profile of 5-FU 0.5%/salicylic acid 10%. BODY.ELECTRONIC SUPPLEMENTARY MATERIAL: Below is the link to the electronic supplementary material. Supplementary material 1 (EPS 1005 kb)

TITLE: Comprehensive Assessment of Coronary Plaque Progression With Advanced Intravascular Imaging, Physiological Measures, and Wall Shear Stress: A Pilot Double‐Blinded Randomized Controlled Clinical Trial of Nebivolol Versus Atenolol in Nonobstructive Coronary Artery Disease ABSTRACT.BACKGROUND: We hypothesized that nebivolol, a β‐blocker with nitric oxide–mediated activity, compared with atenolol, a β‐blocker without such activity, would decrease oxidative stress and improve the effects of endothelial dysfunction and wall shear stress (WSS), thereby reducing atherosclerosis progression and vulnerability in patients with nonobstructive coronary artery disease. ABSTRACT.METHODS AND RESULTS: In this pilot double‐blinded randomized controlled trial, 24 patients treated for 1 year with nebivolol 10 mg versus atenolol 100 mg plus standard medical therapy underwent baseline and follow‐up coronary angiography with assessments of inflammatory and oxidative stress biomarkers, microvascular function, endothelial function, and virtual histology intravascular ultrasound. WSS was calculated from computational fluid dynamics. Virtual histology intravascular ultrasound segments were assessed for vessel volumetrics and remodeling. There was a trend toward more low‐WSS segments in the nebivolol cohort (P=0.06). Low‐WSS regions were associated with greater plaque progression (P<0.0001) and constrictive remodeling (P=0.04); conversely, high‐WSS segments demonstrated plaque regression and excessive expansive remodeling. Nebivolol patients had decreased lumen and vessel areas along with increased plaque area, resulting in more constrictive remodeling (P=0.002). There were no significant differences in biomarker levels, microvascular function, endothelial function, or number of thin‐capped fibroatheromas per vessel. Importantly, after adjusting for β‐blocker, low‐WSS segments remained significantly associated with lumen loss and plaque progression. ABSTRACT.CONCLUSION: Nebivolol, compared with atenolol, was associated with greater plaque progression and constrictive remodeling, likely driven by more low‐WSS segments in the nebivolol arm. Both β‐blockers had similar effects on oxidative stress, microvascular function, and endothelial function. ABSTRACT.CLINICAL TRIAL REGISTRATION: URL: https://clinicaltrials.gov/. Unique identifier: NCT01230892. BODY.INTRODUCTION: Alterations in coronary wall shear stress (WSS) can interact with the endothelium to affect the distribution, progression, and pathophysiology of atherosclerosis,1, 2, 3, 4, 5, 6, 7, 8, 9 and several studies have demonstrated the association between WSS magnitudes and plaque development.3, 4, 5, 6, 7, 8, 9, 10 In particular, low WSS (<1.0 Pa) has been related to plaque progression,5, 6, 7, 8, 9, 10 whereas high WSS (>2.5 Pa) has been associated with increased necrotic core area on virtual histology intravascular ultrasound (VH‐IVUS), a marker of plaque vulnerability.3, 4, 9, 10 The impact of valsartan and simvastatin on the proinflammatory effect of low WSS has been studied in the swine model11; however, the effect of an endothelial protective pharmaceutical agent, such as nebivolol, on the interplay between WSS and the progression of human coronary atherosclerosis has not been investigated. Nebivolol is a third‐generation β1 receptor–selective antagonist with nitric oxide–mediated vasodilatory effect approved by the US Food and Drug Administration for treatment of hypertension and in Europe for left ventricular systolic dysfunction. Previous studies have suggested that nebivolol decreases coronary microvascular resistance through an agonist effect on the endothelial β3 receptor, which mediates nitric oxide– and endothelium‐dependent vasorelaxation.12, 13, 14, 15, 16 Enhanced flow and nitric oxide availability may decrease vascular reactivity and lipid oxidation and prove beneficial in conditions such as atherosclerosis, in which oxidative stress leads to decreased endothelial nitric oxide synthase expression and nitric oxide bioavailability. Potential benefits from nebivolol therapy may be observed in measures of endothelial function, microvascular function, vascular remodeling, or epicardial plaque burden and composition. Accordingly, we conducted a pilot double‐blinded randomized controlled trial on the effect of 1 year of nebivolol versus atenolol therapy on the changes in the number of thin‐capped fibroatheromas (TCFAs) per vessel and in coronary plaque volumetrics among different WSS categories in patients with nonobstructive coronary artery disease. We hypothesized that nebivolol, by decreasing oxidative stress and improving endothelial function, would favorably affect atherosclerosis progression compared with atenolol, a β‐blocker without nitric oxide–mediated activity. BODY.METHODS.PARTICIPANTS AND STUDY DESIGN: This single‐center double‐blinded randomized trial (NCT01230892) enrolled patients with recurrent angina symptoms who presented to the cardiac catheterization laboratory at Emory University Hospital from 2010 to 2012 and were found to have coronary nonobstructive atherosclerotic lesions (<50% stenosis by angiography or <70% stenosis with fractional flow reserve >0.80). Patients provided written informed consent for participation in this study prior to the baseline cardiac catheterization and randomization. Inclusion criteria included patients being on stable medical therapy, presentation of stable angina or non–ST‐segment elevation acute coronary syndrome, and coronary lesion in the proximal 60 mm of an epicardial vessel deemed significant enough by the operator to warrant further evaluation using physiology and VH‐IVUS. Patients were excluded if they presented with cardiogenic shock, ejection fraction <30%, or significant hepatic, hematologic, or renal impairment; had a history of coronary artery bypass surgery or severe valvular heart disease; could not provide informed consent; or had any contraindication to β‐blocker therapy. Anatomically, cases were excluded if there were lesions requiring revascularization or significant visual coronary collaterals. After undergoing baseline coronary angiography with VH‐IVUS and evaluation of coronary microvascular and endothelial function, patients were randomized (1:1) to nebivolol 10 mg daily (first week 5 mg daily) or atenolol 100 mg daily (first week 50 mg daily) for 1 year. Nebivolol 5 mg (Forest Laboratories) and atenolol 50 mg (AstraZeneca Pharmaceuticals) have equivalent efficacy for reducing blood pressure.17 The Investigational Drug Service at our institution performed simple randomization using the online program "Research Randomizer" to generate the randomization plan in blocks of 4; dispensed the blinded study medication (same tablet size, shape, and color, with the same bottles and labels); and maintained separate records using WebIDS, a fully Health Insurance Portability and Accountability Act–compliant system that maintains protocol and drug information, patient profiles, randomization assignments, inventory records, and dispensing and patient return information. Enrolled patients were treated for 12±3 months with the study drug plus standard medical therapy (aspirin, statin, and as needed sublingual nitroglycerin) followed by repeat coronary angiography with VH‐IVUS and assessments of microvascular and endothelial function (Figure 1). Participants, care providers, and study investigators were blinded until after completion of the last follow‐up visit. Figure 1Study flowchart. The Emory University institutional review board approved the study, and there were no significant changes to the protocol after the trial commenced. The primary end point was the change in the number of TCFAs per vessel between baseline and follow‐up. Secondary end points included inflammatory and oxidative stress biomarkers, endothelial function, microvascular function, static and serial arterial remodeling, and VH‐IVUS plaque area and composition at baseline and 1‐year follow‐up. BODY.METHODS.LIPIDS AND BIOMARKERS: Basic metabolic panel, complete blood count, fasting lipid panel, and C‐reactive protein level were collected the day of angiography and measured by the hospital medical laboratory services. Samples for plasma glutathione and cystine measurements were collected immediately after obtaining vascular access in the cardiac catheterization laboratory, were frozen at −80°C, and were measured by the Emory University clinical biomarkers core laboratory. These samples have been shown to be stable under these conditions for 1 year.18 BODY.METHODS.CARDIAC CATHETERIZATION WITH ASSESSMENT OF ENDOTHELIAL AND MICROVASCULAR FUNCTION: All ß‐blockers were withheld for ≥48 hours and long‐acting nitrates were withheld for ≥24 hours prior to cardiac catheterization (both baseline and follow‐up procedures). Procedures were performed in the morning after patients had fasted for at least 8 hours. Systolic and diastolic blood pressures and heart rate were measured prior to the start of the procedure. Patients underwent angiography in a biplane cardiac catheterization system (Toshiba America Medical Systems) using a standard 6F technique. Pressure and velocity measurements were obtained using a 0.014‐in pressure and Doppler flow velocity monitoring guidewire (ComboWire: Volcano Corporation). For safety considerations, only the left coronary system was interrogated. The ComboWire was advanced to the guide catheter tip, at which the aortic pressure and guidewire pressures were equalized. Microvascular function was evaluated from pressure and velocity responses to intravenous adenosine infusion (140 μg/kg per minute) for 3 minutes. At maximal hyperemia, fractional flow reserve was measured as the ratio of distal to aortic pressure, and hyperemic microvascular resistance was measured as the ratio of distal pressure to average peak flow velocity. Coronary flow velocity reserve was defined as the ratio of hyperemic to basal average peak flow velocity. Velocity measurements demonstrated good reproducibility, with a concordance correlation coefficient of 0.979 (95% CI 0.966–0.988).9 Endothelial function was assessed as the percentage change in coronary diameter and blood flow in response to intracoronary acetylcholine (off‐label use) using Doppler flow velocity measurements and quantitative coronary angiography (QAngio XA 7.3; Medis Medical Imaging Systems). Patients were first evaluated with 10−8mol/L acetylcholine for safety prior to proceeding with 10−6mol/L acetylcholine. Figure 2 demonstrates Doppler velocity envelopes prior to and during medication infusion in 1 patient at baseline and follow‐up. Figure 2Doppler velocity waveforms obtained from a patient undergoing endothelial and microvascular function assessment at baseline and 1‐year follow‐up. APV indicates average peak velocity; CFR, coronary flow reserve; IC, intracoronary; IV, intravenous. BODY.METHODS.VH‐IVUS ACQUISITION AND ANALYSIS: VH‐IVUS acquisition was performed after administration of 200 μg intracoronary nitroglycerin using phased‐array 20 MHz Eagle Eye catheters and a s5 Imaging System (Volcano Corporation). Automated motorized pullback (0.5 mm/s) was performed, and VH‐IVUS images were continuously acquired up to the guide catheter in the aorta (up to 60 mm of the proximal vessel). Angiography was used to record the catheter start position and its relationship to anatomic branching landmarks to aid with coregistration. Offline analysis was performed at the Emory cardiovascular imaging and biomechanical core laboratory by experienced investigators who were blinded to the patients' clinical data according to the criteria of the American College of Cardiology Clinical Consensus document on IVUS,19, 20 using VIAS version 3.0 (Volcano Corporation) and echoPlaque 4.0.27 (INDEC Medical Systems). Measurements of the external elastic membrane (EEM), plaque, and lumen cross‐sectional areas were performed for every recorded VH‐IVUS cross‐section (0.5‐mm thickness), defined as a segment in the current analysis. Plaque burden was calculated as plaque area divided by EEM area. Segments involving bifurcating branch points or precluding complete lumen or vessel wall planimetry were excluded from analysis. Intraobserver analysis demonstrated good reproducibility for plaque area (concordance correlation coefficient 0.968 [95% CI 0.965–0.971]).9 Different nomenclatures have been used to describe arterial remodeling patterns. We defined static remodeling as the ratio of lesion to reference EEM area, also known as the remodeling index. We also used excessive expansive, compensatory, and constrictive to describe 3 patterns of serial remodeling.19 For each segment, positive ΔEEM area was defined as positive remodeling, and negative ΔEEM area was defined as constrictive remodeling. Segments with positive remodeling were further subdivided as compensatory if the ratio of ΔEEM area to Δplaque area was between 0.0 and 1.0 or as excessive expansive otherwise. Finally, lesions were classified based on plaque composition as assessed by VH‐IVUS (fibrous, fibrofatty, necrotic core, and dense calcium) for each segment.20, 21 Fibroatheromas were defined as ≥40% plaque burden and ≥10% necrotic core; TCFAs were fibroatheromas with necrotic core abutting the lumen in at least 3 consecutive frames. BODY.METHODS.COMPUTATIONAL FLUID DYNAMICS AND BASELINE WSS ANALYSIS: WSS analysis methodology using the ANGUS method has been described previously.1, 9 Briefly, the 3‐dimensional path of the VH‐IVUS catheter was determined using corresponding biplane angiographic projections acquired at the start of pullback. Each frame was rotated and aligned perpendicular to the catheter core to reconstruct the main artery of interest. Arterial branches were added based on information from angiography and VH‐IVUS. Patient‐specific pulsatile inlet boundary conditions were determined from Doppler wire‐derived waveforms, and the outlets were assumed to be pressure‐free. The reconstructed surface was meshed and imported into Fluent (ANSYS). After computing the pulsatile flow field in the region of interest, WSS was determined as a function of time in the cardiac cycle and spatial location around the lumen (Video S1, Figure 3) and then averaged over time and circumference at each cross‐section for quantitative analysis. Although WSS is a continuous and dynamic variable, based on previous cell culture and experimental and human data, WSS magnitudes were categorized as low (<1.0 Pa), intermediate (≥1.0 and <2.5 Pa), or high (≥2.5 Pa).3, 9, 22, 23 Figure 3A patient‐specific WSS profile of the left anterior descending coronary artery demonstrates areas of variable WSS magnitudes. WSS indicate wall shear stress. BODY.METHODS.STATISTICAL ANALYSIS: Sample size was not determined strictly from effect sizes because the purpose of this pilot study was to generate data to power future clinical trials. It was estimated using previous comparative studies between nebivolol and atenolol on endothelial and microvascular function assuming a 0.05 significance level and 0.80 power value.13, 15, 16 Continuous variables are described as mean±SD or median and interquartile range, as appropriate, and categorical variables are described as counts and proportions. For patient‐level analyses, the chi‐square statistic was used to compare categorical variables (eg, hypertension), and the 2‐sample t test was used for continuous variables (eg, age). The differences between baseline and follow‐up patient‐level measures (eg, systolic blood pressure) were computed for each group, and the differences between the 2 groups were compared with the 2‐sample t test. Correlated error is introduced by the clustering of arterial segments within patients. To correct for this, P values were adjusted with linear regression for continuous outcomes (eg, Δlumen area) and ordered logistic regression for categorical variables (eg, serial arterial remodeling). In both situations, the Huber White Sandwich Estimator was used to adjust the P values for correlated error.24, 25 To further examine the association of WSS, drug, and change measures, mixed models with WSS and drug as predictor variables were run. Although there are many observations at the VH‐IVUS segmental level, the effective sample size is 24 participants, and 2 covariates were determined to be a reasonable limit. Analyses were performed using SAS 9.3 (SAS Institute) or Stata 13.1 (StataCorp LP). P<0.05 was established as the level of statistical significance. BODY.RESULTS.STUDY PARTICIPANTS, ENTIRE COHORT: Fifty patients consented to study participation prior to baseline coronary angiography. Two patients who underwent initial cardiac catheterization developed severe vasospasm during 10−6mol/L acetylcholine infusion. They were successfully treated during the procedure and did well clinically but did not participate further in this trial or undergo randomization. Another 19 patients did not meet angiographic inclusion criteria and were considered screen failures. Consequently, 29 patients were enrolled in the study and underwent randomization. A total of 25 patients returned for follow‐up; 1 case was excluded from analysis due to poor data quality (Figure 1). For the remaining 24 patients, physiological interrogation was performed in the left circumflex artery for 1 patient and in the left anterior descending artery for 23 patients. Table 1 displays baseline patient characteristics. At follow‐up, both systolic and diastolic blood pressures decreased numerically (P value not significant), but heart rate changed significantly by −5 beats per minute (interquartile range −11 to +1 beats per minute; P=0.02). Low‐density lipoprotein cholesterol level also increased by 10 mg/dL (interquartile range −3 to +29 mg/dL; P=0.04). There were no significant changes in coronary physiology or endothelial function between baseline and follow‐up (Table 2). Table 1 Patient Demographics and Characteristics Total (n=24) Atenolol (n=12) Nebivolol (n=12) P Value Age, y 52.5±10.3 49.8±10.7 55.2±9.5 0.21 Male 9 (38) 5 (42) 4 (33) 0.67 White race 18 (75) 8 (67) 10 (83) 0.35 Body mass index 29.1±5.8 30.0±6.4 28.2±5.2 0.46 Follow‐up time period, months 12.4±1.0 12.5±0.6 12.4±1.3 0.81 Cardiovascular risk factors Hypertension 16 (67) 9 (75) 7 (58) 0.39 Diabetes mellitus 4 (17) 2 (17) 2 (17) 1.00 Dyslipidemia 18 (75) 8 (67) 10 (83) 0.35 Prior myocardial infarction 4 (17) 2 (17) 2 (17) 1.00 Smoking history 12 (50) 7 (58) 5 (42) 0.41 Family history of coronary disease 11 (46) 4 (33) 7 (58) 0.22 Presentation Non–ST‐segment elevation myocardial infarction 1 (4) 1 (8) 0 (0) Unstable angina 5 (21) 2 (17) 3 (25) 0.52 Stable angina 17 (71) 8 (67) 9 (75) Medication use Aspirin 20 (83) 10 (83) 10 (83) 1.00 P2Y 12 inhibitor 6 (25) 3 (25) 3 (25) 1.00 Statin 23 (96) 12 (100) 11 (92) 0.31 Calcium channel blocker 11 (46) 7 (58) 4 (33) 0.22 Long‐acting nitrate 17 (71) 10 (83) 7 (58) 0.18 ACE inhibitor or ARB 6 (25) 3 (25) 3 (25) 1.00 Values are expressed as n (%) or mean±SD. ACE indicates angiotensin‐converting enzyme; ARB, angiotensin II receptor blocker. Table 2 Patient and Vessel Characteristics at Baseline and Change After 1 Year Baseline Change After 1 Year Atenolol (n=12) Nebivolol (n=12) P Value Atenolol (n=12) Nebivolol (n=12) P Value Vital signs Systolic BP, mm Hg 137 [122, 145] 143 [122, 159] 0.51 −8 [−25, 10] −7 [−20, 1] 0.95 Diastolic BP, mm Hg 73 [71, 85] 78 [69, 92] 0.63 0 [−8, 14] −8 [−14, 6] 0.08 Heart rate, bpm 71 [60, 85] 68 [62, 78] 0.91 −5 [−11, 6] −6 [−11, −1] a 0.66 Fasting lipid panel Total cholesterol, mg/dL 158 [136, 171] 149 [119, 160] 0.13 15 [−1, 31] 14 [−2, 55] 0.80 HDL cholesterol, mg/dL 48 [38, 54] 44 [43, 54] 0.75 3 [−5, 16] −3 [−8, 2] 0.16 LDL cholesterol, mg/dL 87 [69, 104] 81 [72, 115] 0.52 6 [−3, 29] 13 [−4, 35] 0.89 Markers of inflammation and oxidative stress C‐reactive protein, mg/L 1.87 [0.50, 3.82] 2.05 [1.30, 4.06] 0.48 0.33 [−1.72, 2.48] 0.07 [−0.78, 0.42] 0.72 Cystine, μmol/L 84 [72, 112] 88 [79, 108] 0.41 10 [−4, 20] 17 [−7, 43] 0.37 Glutathione, μmol/L 1.04 [0.87, 1.60] 0.98 [0.79, 1.26] 0.74 −0.13 [−0.56, 0.46] −0.01 [−0.04, 0.17] 0.52 Cystine/glutathione ratio 84 [63, 100] 88 [69, 139] 0.53 31 [−2, 44] 15 [−24, 48] 0.78 Physiology, endothelial function, and vessel characteristics FFR 0.98 [0.94. 1.00] 0.91 [0.90, 0.98] 0.08 −0.02 [−0.08, 0.01] −0.01 [−0.03, 0.05] 0.39 CFR 2.07 [1.82, 2.38] 1.87 [1.62, 2.33] 0.74 0.41 [0.06, 0.48] 0.71 [−0.58, 1.26] 0.97 HMR 1.75 [1.47, 2.66] 1.91 [1.46, 2.21] 0.89 0.07 [−0.54, 1.07] −0.06 [−0.24, 0.43] 1.00 %Diameter change to ACh −5 [−22, 3] −2 [−8, 15] 0.47 2 [−11, 30] 3 [−12, 25] 0.89 %CBF change to ACh 60 [−11, 225] 142 [37, 181] 0.81 −5 [−172, 121] 87 [30, 385] 0.47 TCFAs per patient 2.2±1.2 1.6±1.0 0.27 −0.4±0.8 −0.3±1.0 0.94 Segment characteristics VH‐IVUS run length, mm 54.3±10.9 62.5±21.1 0.25 Minimum lumen area, mm 2 6.11 [3.85, 7.15] 4.15 [3.87, 7.77] 0.93 Static remodeling 0.60±0.19 0.58±0.22 0.50 EEM area, mm 2 16.5±7.3 17.5±7.3 0.76 0.20±1.99 a −0.78±2.46 a 0.02 Lumen area, mm 2 10.9±4.5 12.7±5.9 0.41 0.33±2.08 a −1.22±2.68 a 0.004 Plaque area, mm 2 5.7±4.8 4.9±3.3 0.66 −0.13±2.44 a 0.44±1.95 a 0.27 FI area, mm 2 1.67±2.86 1.08±1.66 0.51 −0.16±1.58 a 0.14±1.02 a 0.26 FF area, mm 2 0.23±0.46 0.16±0.35 0.57 −0.04±0.38 a 0.02±0.38 a 0.27 NC area, mm 2 0.48±1.07 0.36±0.80 0.77 −0.07±0.76 a 0.03±0.49 a 0.35 DC area, mm 2 0.09±0.17 0.15±0.38 0.28 0.01±0.17 a 0.04±0.24 a 0.34 Values are expressed as mean±SD or median [IQR]. ACh indicates acetylcholine; BP, blood pressure; bpm, beats per minute; CBF, coronary blood flow; CFR, coronary flow reserve; DC indicates dense calcium; EEM, external elastic membrane; FF, fibrofatty; FFR, fractional flow reserve; FI, fibrous; HDL, high‐density lipoprotein; HMR, hyperemic myocardial resistance; LDL, low‐density lipoprotein; NC, necrotic core; RI, remodeling index; TCFAs, thin‐capped fibroatheromas; VH‐IVUS, virtual histology intravascular ultrasound. a Significant change from baseline ( P <0.05). Baseline mean VH‐IVUS run length was 58±17 mm, lumen area was 11.84±5.38 mm2, EEM area was 17.08±7.35 mm2, and plaque area was 5.24±4.07 mm2. At follow‐up, the change in EEM area was −0.33±2.31 mm2 (P<0.0001), in lumen area was −0.51±2.54 mm2 (P<0.0001), in plaque area was 0.18±2.21 mm2 (P<0.0001), in fibrous area was −0.01±1.33 mm2 (P=0.67), in fibrofatty area was −0.01±0.38 mm2 (P=0.22), in necrotic core area was −0.02±0.64 mm2 (P=0.07), and in dense calcium area was 0.03±0.21 mm2 (P<0.0001). BODY.RESULTS.RELATIONSHIP BETWEEN BASELINE CORONARY WSS, SERIAL ARTERIAL REMODELING, AND PLAQUE PROGRESSION: Coronary WSS was calculated in 1843 VH‐IVUS segments using computational fluid dynamics modeling. There were 428 (23%) low‐, 920 (50%) intermediate‐, and 495 (27%) high‐WSS segments. At follow‐up, low‐WSS segments demonstrated an increase in plaque area (P<0.0001) and a decrease in lumen area (P<0.0001) compared with intermediate‐ and high‐WSS segments (Table 3) and more constrictive remodeling (P=0.04) (Figure 4). Table 3 Comparison of the Change in Virtual Histology Intravascular Ultrasound Measures After 1 year by Baseline WSS Category Characteristic Low WSS (n=428) Intermediate WSS (n=920) High WSS (n=495) P Value ΔLumen area, mm 2 −1.17±1.24 −0.64±1.61 0.61±2.15 <0.0001 ΔEEM area, mm 2 −0.70±1.32 −0.37±1.29 0.05±1.82 0.40 ΔPlaque area, mm 2 0.47±1.37 0.27±1.82 −0.56±2.77 <0.0001 ΔFibrous area, mm 2 0.21±0.76 0.02±1.08 −0.47±1.64 <0.0001 ΔFibrofatty area, mm 2 0.04±0.30 −0.01±0.24 −0.05±0.35 <0.0001 ΔNecrotic core area, mm 2 −0.07±0.71 −0.01±0.66 −0.17±0.88 <0.0001 ΔDense calcium area, mm 2 −0.01±0.22 0.04±0.22 0.04±0.26 <0.0001 Values are expressed as mean±SD. EEM indicates external elastic membrane; WSS, wall shear stress. Figure 4Serial arterial remodeling by baseline WSS categories at 1 year follow up. P value is for comparison of frequency of 3 remodeling groups across 3 WSS categories. WSS indicate wall shear stress. BODY.RESULTS.COMPARISONS BETWEEN NEBIVOLOL AND ATENOLOL: Between the 2 cohorts, there were no significant differences in clinical characteristics or cardiac medications (Table 1) or other baseline and follow‐up variables such as cholesterol levels, inflammatory and oxidative stress biomarkers, coronary flow velocity reserve, hyperemic microvascular resistance, or coronary endothelial function (Table 2). There was a trend of more segments in the nebivolol cohort with low WSS (317 versus 111) and fewer segments with high WSS (175 versus 320) compared with the atenolol arm (Figure 5). Figure 5Distribution of baseline WSS by β‐blocker. P value is for comparison of the frequency of 3 WSS categories across 2 β‐blockers. WSS indicates wall shear stress. On VH‐IVUS, nebivolol segments demonstrated significantly decreased EEM area (P=0.02) and lumen area (P=0.004) but similar changes in plaque components and number of TCFAs per vessel compared with atenolol (Table 2). Nebivolol also showed increased plaque area at follow‐up (P<0.001), whereas atenolol demonstrated plaque regression (P=0.047); however, the difference between the 2 arms was not significant (P=0.27). With respect to arterial remodeling, static remodeling (remodeling index) was similar between the 2 groups, but serial remodeling was significantly different (P=0.002), with nebivolol segments demonstrating more constrictive remodeling (73% versus 46%) and less excessive expansive remodeling (16% versus 39%) than atenolol (Figure 6). Figure 6Serial arterial remodeling by β‐blocker at 1‐year follow‐up. P value is for comparison of the frequency of 3 remodeling groups across 2 β‐blocker categories. These observations could have been related to differences in baseline WSS patterns between the arms or to pharmacological therapy with the 2 different β‐blockers, so VH‐IVUS results were stratified by categories of WSS (Table 4) and β‐blocker (Table 5). Among low‐WSS segments (Table 4), those treated with nebivolol demonstrated decreased ΔEEM area compared with atenolol (P=0.02), consistent with a higher percentage of constrictive serial remodeling (83% versus 63%). Table 4 Comparison of Change in Virtual Histology Intravascular Ultrasound Measures by β‐Blocker, Stratified by Baseline Wall Shear Stress Categories Low WSS Intermediate WSS High WSS Atenolol (n=111) Nebivolol (n=317) P Value Atenolol (n=382) Nebivolol (n=538) P Value Atenolol (n=320) Nebivolol (n=175) P Value ΔLumen area, mm 2 −0.80±1.15 −1.29±1.25 0.22 0.21±1.79 −1.24±1.15 0.002 1.15±2.38 −0.39±1.08 0.02 ΔEEM area, mm 2 0.01±1.06 −0.95±1.31 0.02 −0.02±1.53 −0.62±1.02 0.15 0.29±1.96 −0.40±1.44 0.43 ΔPlaque area, mm 2 0.81±1.05 0.35±1.45 0.30 −0.23±2.38 0.62±1.15 0.28 −0.86±3.23 −0.01±1.45 0.30 ΔFI area, mm 2 0.36±0.84 0.16±0.72 0.23 −0.18±1.44 0.16±0.68 0.40 −0.66±1.94 −0.13±0.79 0.31 ΔFF area, mm 2 0.04±0.23 0.03±0.32 0.47 −0.03±0.25 0.00±0.24 0.77 −0.09±0.38 0.01±0.27 0.34 ΔNC area, mm 2 0.07±0.34 −0.12±0.79 0.45 −0.15±0.92 0.09±0.35 0.14 −0.25±1.06 −0.01±0.30 0.25 ΔDC area, mm 2 −0.01±0.12 −0.01±0.24 0.78 0.01±0.16 0.06±0.25 0.19 0.02±0.22 0.09±0.31 0.15 P ‐values are for comparison between 2 β‐blocker categories. DC indicates dense calcium; EEM, external elastic membrane; FF, fibrofatty; FI, fibrous; NC, necrotic core; WSS, wall shear stress. Table 5 compares VH‐IVUS changes by WSS categories within each β‐blocker cohort. Compared with high‐WSS segments, low‐WSS segments demonstrated greater decrease in lumen area (P<0.0001) and increase in plaque area (P<0.01). After adjustment for β‐blocker, WSS category remained an independent predictor of changes in lumen area (P<0.0001) and plaque area (P<0.0001). Table 5 Comparison of Change in VH‐IVUS Measures by Baseline WSS Categories, Stratified by Type of β‐Blocker Atenolol Nebivolol Low WSS (n=111) Intermediate WSS (n=382) High WSS (n=320) P Value Low WSS (n=317) Intermediate WSS (n=538) High WSS (n=175) P Value ΔLumen area, mm 2 −0.80±1.15 0.21±1.79 1.15±2.38 <0.0001 −1.29±1.25 −1.24±1.15 −0.39±1.08 <0.0001 ΔVessel area, mm 2 0.01±1.06 −0.02±1.53 0.29±1.96 0.002 −0.95±1.31 −0.62±1.02 −0.40±1.44 <0.0001 ΔPlaque area, mm 2 0.81±1.05 −0.23±2.38 −0.86±3.23 <0.0001 0.35±1.45 0.62±1.15 −0.01±1.45 0.006 ΔFI area, mm 2 0.36±0.84 −0.18±1.44 −0.66±1.94 <0.0001 0.16±0.72 0.16±0.68 −0.13±0.79 <0.0001 ΔFF area, mm 2 0.04±0.23 −0.03±0.25 −0.09±0.38 <0.0001 0.03±0.32 0.00±0.24 0.01±0.27 0.02 ΔNC area, mm 2 0.07±0.34 −0.15±0.92 −0.25±1.06 <0.0001 −0.12±0.79 0.09±0.35 −0.01±0.30 0.01 ΔDC area, mm 2 −0.01±0.12 0.01±0.16 0.02±0.22 0.57 −0.01±0.24 0.06±0.25 0.09±0.31 <0.0001 P ‐values are for comparison among 3 WSS categories. DC indicates dense calcium; FF, fibro‐fatty; FI, fibrous; NC, necrotic core; WSS, wall shear stress. BODY.DISCUSSION: This investigation is the first randomized controlled clinical trial studying the impact of a β1‐blocker with endothelial β3‐receptor agonist activity on the proatherosclerotic effect of coronary WSS. The major observations were as follows: First, there was greater plaque progression (P<0.0001) and constrictive remodeling in segments with low WSS (P=0.04) and, conversely, greater plaque regression and excessive expansive remodeling in segments with high WSS. Second, there were greater reductions in lumen area (P=0.004) and vessel area (P=0.02), resulting in more constrictive remodeling, with nebivolol (P=0.002), likely driven by a trend toward more low‐WSS segments in the nebivolol cohort (P=0.06). Third, after adjusting for the type of β‐blocker therapy, low‐WSS segments remained significantly associated with lumen loss and plaque progression (P<0.0001). Fourth, there were no significant differences in oxidative stress or inflammatory biomarker levels, endothelial function, microvascular function, or number of TCFAs per vessel between nebivolol and atenolol. Coronary atherosclerosis pathophysiology operates through complex multitude of pathways, including endothelial dysfunction as a conduit for systemic risk factors to affect the vasculature.18, 26, 27 The considerable number of investigations into the effects of WSS magnitudes on plaque development emphasizes this complexity. Low WSS can promote atherogenesis by changing endothelial cell morphology,2 enhancing production of reactive oxygen species and inflammatory molecules,22 and stimulating vascular smooth muscle cell migration.8, 28 In our study, we observed that low‐WSS regions were associated with more constrictive remodeling (ΔEEM −0.70 mm2, P=0.04) and an absolute increase in plaque area of 0.47 mm2 (P<0.0001). Conversely, high WSS can also contribute to plaque destabilization.4, 10, 29, 30, 31 Histological studies have implicated high WSS in smooth muscle cell apoptosis and proteoglycan matrix degradation,29 whereas animal studies have demonstrated increased mechanical strain, vascular inflammation, macrophage activity, and expansive remodeling in high‐WSS regions.30, 32 We previously observed in human coronary arteries that, compared with intermediate‐WSS segments, high‐WSS segments trended toward more excessive expansive arterial remodeling,9, 23 an exaggerated remodeling pattern in which both vessel and lumen dimensions increase proportionally more than plaque area. Although this type of remodeling in high‐WSS segments may be partially explained by adaptive arterial enlargement in an attempt to restore WSS to a more physiological level,31 it can also drive plaque progression through continued lipid accumulation and inflammation. In this study, we demonstrated again that high‐WSS regions were compensated by excessive expansive remodeling (Figure 5) with fibrous and fibrofatty tissue regression (Table 3). The observation that nebivolol patients had more constrictive remodeling and lumen loss compared with atenolol patients in this study was interesting. Although this was a double‐blinded randomized controlled trial, the nebivolol cohort had a numerically greater number of low coronary WSS segments per patient compared with the atenolol cohort (26.4 versus 9.3, P=0.06), despite having similar baseline plaque areas (4.9 mm2 versus 5.7 mm2, P=0.66), static vascular remodeling (remodeling index 0.58 versus 0.60, P=0.50), and coronary flow reserve (1.87 versus 2.07, P=0.74). After adjusting for type of β‐blocker, low WSS remained significantly associated with plaque progression and lumen loss (both P<0.0001), suggesting that WSS and not type of β‐blocker therapy was driving these findings. Interestingly, in our study, nebivolol and atenolol had similar changes in lipid profiles, inflammation and oxidative stress biomarkers, and coronary endothelial and microvascular function. In another study of hypertensive patients, we also observed no differences between nebivolol and metoprolol regarding pulse wave velocity, a noninvasive measurement of arterial stiffness and endothelial function, or oxidative stress biomarker levels.33 In addition, there were no significant differences between nebivolol and atenolol with respect to necrotic core area or the number of TCFAs per vessel. Taken together, the current investigation demonstrates no significant favorable or adverse effects of nebivolol compared with atenolol on comprehensive atherosclerosis phenotyping. Despite the multidimensional nature of atherosclerosis pathophysiology, previous mechanistic investigations into the impact of pharmacotherapies on atherosclerosis development have generally relied on basic measures of plaque assessment such as the change in carotid intima media thickness34, 35 or coronary atheroma volume.36, 37, 38 These measures may underestimate the effect of antiatherosclerotic therapies on the multiple known atherosclerotic pathways and processes. To our knowledge, no prior investigation has comprehensively examined the incremental effect of a potential antiatherosclerotic therapy on epicardial and microvascular endothelial‐dependent and ‐independent function, atherosclerosis burden and composition, static and serial arterial remodeling, and oxidative stress and inflammatory pathways in the context of coronary WSS. This investigation offers a blueprint for how novel cardiovascular therapies can be evaluated, given the numerous possible mechanisms for therapeutic intervention. BODY.DISCUSSION.LIMITATIONS: The study was powered for VH‐IVUS end points but may not have been adequately powered to detect changes in endothelial and microvascular function or number of TCFAs per artery. Although the nebivolol dose used in this trial was approved for clinical use, the effective concentration may be too low or the duration of follow‐up insufficient to affect the prespecified end points. Nevertheless, this study is the most comprehensive atherosclerosis phenotyping double‐blinded randomized controlled trial performed thus far and can establish the methodology for future coronary atherosclerosis trials for novel pharmaceutical agents such as protein convertase subtilisin/kexin type 9 inhibitors. Plaque composition data are derived from VH‐IVUS, which has inherent limitations compared with histology but can be performed in vivo in coronary arteries and has been validated by several studies.6, 7, 8, 9, 20, 21 In addition, although there were many arterial segments, the effective sample size was 24 participants; this limits the number of covariates used in multivariate analysis. Finally, this was a randomized clinical trial between nebivolol and atenolol; however, the constrictive remodeling seen in the nebivolol arm is likely related to the greater number of baseline segments with low WSS. Although randomization can be readily performed with prespecified balance in demographics, it is currently impractical, given the time‐ and resource‐intensive nature of computational fluid dynamic modeling, to randomize patients based on their distribution of coronary WSS segments. BODY.CONCLUSIONS: Compared with atenolol, nebivolol demonstrated greater luminal reduction and constrictive remodeling, but this was likely driven by the higher number of low‐WSS segments in the nebivolol cohort. There were no significant differences in coronary endothelial function or microvascular function between treatment groups. This study suggests that any impact of an endothelial β3‐receptor agonist on the effects of WSS magnitudes or on the natural history of epicardial and microvascular atherosclerosis, if present, is minimal. Importantly, it lays down the methodological framework for future mechanistic evaluation of pharmacotherapy on human coronary atherosclerosis. BODY.DISCLOSURES: McDaniel consults for Medicure, Inc. Samady receives research funding from Forest Laboratories an Allergan Affiliate, Volcano Corporation, St. Jude Medical, Gilead Sciences, Medtronic, Inc and Abbott Vascular. BODY.SUPPORTING INFORMATION: Video S1. A patient‐specific wall shear stress (WSS) profile of the left anterior descending coronary artery demonstrates areas of variable WSS magnitudes throughout the cardiac cycle.Click here for additional data file.

TITLE: Readiness to Be Physically Active and Self-Reported Physical Activity in Low-Income Latinas, California WISEWOMAN, 2006-2007 ABSTRACT.INTRODUCTION: Latinas are more likely to be inactive than non-Hispanic white women. Although 74% of Latinas report no leisure-time activity, few interventions have been designed to promote physical activity among these women. The objective of this study was to assess the effect of the California WISEWOMAN program on low-income Latinas's readiness to change physical activity and on self-reported physical activity behaviors. ABSTRACT.METHODS: We screened 1,332 women for cardiovascular disease risk factors and randomly assigned 1,093 women to 2 groups: an enhanced intervention (n = 552) or usual care (n = 541). The enhanced intervention was delivered by community health workers in one-on-one counseling sessions. We examined self-reported readiness to change and physical activity at baseline and 12-month follow-up among participants who completed both assessments (n = 868). ABSTRACT.RESULTS: Mean age of participants was 52 years (standard deviation, 6 y); most (65%) were Mexican or Mexican American, and most (81%) were not high school graduates. A higher percentage (67%) of the enhanced intervention group was in the action/maintenance stage for vigorous physical activity at follow-up compared with baseline (47%). We found no such change among women in usual care (52%, baseline; 58%, follow-up). A higher percentage of the enhanced intervention group also reported significant increases in moderate (71%, baseline; 84%, follow-up) and vigorous (13% to 33%) physical activity at follow-up than at baseline. Women in usual care reported no changes. ABSTRACT.CONCLUSION: A culturally tailored adaptation of the WISEWOMAN program that used community health workers significantly improved both self-reported readiness to engage in physical activity and vigorous physical activity among low-income Latinas. BODY.INTRODUCTION: Although death from cardiovascular disease (CVD) has declined in recent decades, disparities exist for low-income Latinas (1,2). These disparities may be due to high rates of obesity and low levels of physical activity. Latinas are more likely to be inactive than non-Hispanic white women (2); 74% of Latinas report no physically active leisure-time activity (3). Despite the obvious public health need, few interventions have been designed to promote physical activity among Latinas. A recent review of the literature found only 12 intervention studies that promoted physical activity in Latinas; only 4 showed significant increases in physical activity, and few had any long-term follow-up to determine maintenance of physical activity changes (4). Many of these interventions may have failed because they had a poor foundation in behavior-change theory, especially as the theory relates to the cultural and contextual factors that determine behavior in Latinas (4). To address this limitation, the California Department of Public Health (CDPH) tested a cultural adaptation for Latinas of a successful program used to promote healthy behaviors in African American women who have CVD (5-7). The program was created by the University of North Carolina at Chapel Hill (UNC-Chapel Hill) for use by the North Carolina Well-Integrated Screening and Evaluation for Women Across the Nation (WISEWOMAN) program (8). Behavior-change theory, especially as interpreted through the transtheoretical and socioecological models (9), was a critical foundation of the original program and of our adaptation. All aspects of the California program were delivered by bicultural, bilingual community health workers (CHWs) (8). The program resulted in improvements in the CVD risk profile, as measured by the 10-year coronary heart disease risk, of women who participated in the intervention group compared with women in usual care (10). The objective of this study was to assess the effect of the California WISEWOMAN program on participants'readiness to change 4 physical activity behaviors (engaging in vigorous physical activity, taking up new physical activities, doing daily activities more briskly, and changing daily habits to be more active) and self-reported physical activity. We measured readiness to change to verify the theoretical foundations of the intervention and to study how changing readiness to engage in behavior was related to self-reported behavior change. BODY.METHODS.STUDY DESIGN: Study design was a within-site randomized intervention of 2 study groups (enhanced intervention group [EIG] and usual care group [UCG]) from January 2006 to June 2007. Details on study design, eligibility, recruitment, enrollment, and initial study outcomes are described elsewhere (8,10). The California WISEWOMAN study protocol was approved by the Centers for Disease Control and Prevention (CDC) and the California Committee for the Protection of Human Subjects institutional review boards. BODY.METHODS.STUDY SETTINGS: Sites for both the EIG and UCG were community clinics that were participating in the California National Breast and Cervical Cancer Early Detection Program (NBCCEDP) and serving a large population of low-income, Latino patients. We selected the 4 clinics (2 in San Diego and 2 in Los Angeles counties) in 2005 through a competitive application process. The mission of these clinics was to address the health concerns of primarily Hispanic/Latino low-income communities; health care providers spoke both Spanish and English. BODY.METHODS.ELIGIBILITY, RECRUITMENT, AND ENROLLMENT: We screened 1,332 Latinas for eligibility; 1,093 women met the inclusion criteria (8,10). Women were recruited through the NBCCEDP by telephone calls, cards and letters, and face-to-face invitations during NBCCEDP appointments. To be eligible, women were required to self-identify as Hispanic or Latina, speak English or Spanish, be aged 40 to 64 years, have an annual income at or below 200% of the federal poverty level, have inadequate or no health insurance, have a blood pressure or total cholesterol level considered at risk for developing CVD (either ≥120 mm Hg systolic or ≥80 mm Hg diastolic for blood pressure or currently taking medications to lower blood pressure and either ≥200 mg/dL for total cholesterol or taking medications to lower cholesterol). Women were excluded if they were pregnant or planning to become pregnant during the study period, if they had current or past history of a CVD event or condition, or if they had a blood pressure measurement of 180/110 mm Hg or more or a cholesterol measurement of 400 mg/dL or more. Women with these high values were immediately referred for follow-up care. Eligibility was determined by a nurse. After baseline assessment and clinical measurements, the clinic study team nurse randomly assigned eligible participants to the EIG (n = 552) or UCG (n = 541). We enrolled participants between January 2006 and August 2006. BODY.METHODS.INTERVENTION: Enhanced intervention. The enhanced intervention was delivered by CHWs as 3 individually tailored, one-on-one counseling sessions. The sessions occurred approximately 1 month, 2 months, and 6 months after screening. Each session averaged 50 minutes. Participants received transportation tokens and grocery store vouchers as incentives for attendance. Adaptation of the WISEWOMAN curriculum for our participants is detailed elsewhere (11) and followed expert recommendations (12). In short, to adapt the program, investigators at UNC-Chapel Hill supervised an expert panel review, conducted focus groups, translated the program to Spanish, elicited feedback on the Spanish version, back-translated the new version to English, and reviewed the back-translation to ensure fidelity to the original program. Vida Saludable, Corazón Contento! is available from the Center of Excellence for Training and Research Translation (5). The curriculum emphasized 6 core elements: assessment, tailored feedback, goal setting, guidelines and strategies to overcome barriers, follow-up and reinforcement, and social support. It also used theoretical concepts, such as self-efficacy, self-regulation through self-monitoring, and readiness for change. We tailored the program for each participant by using their self-identified barriers and facilitators to behavior change and emphasizing contextual factors important to Latinas, such as family and social support (4). We also allowed women to choose different activities to meet their goals as long as they were at least of moderate intensity. We visited each study clinic site twice during the study period to ensure fidelity to the study protocol. We determined whether all program elements were present, observed screening and counseling sessions, assessed accuracy of medical record chart abstraction, and held discussions with site staff and administrators. We used monthly teleconferences for similar purposes. Because CHWs delivered the enhanced intervention and collected data on self-reported behavior, we emphasized the importance of avoiding bias in data collection. Usual care. UCG participants received usual care for elevated blood pressure or cholesterol from each clinic site. EIG participants had the same access to usual care. Usual care may have included any or all of the following: healthy behavior education by a provider, distribution of healthy lifestyle handouts, and referral of women to healthy lifestyle education classes if available at the clinic site. Care was provided by physicians and nurses in Spanish and English at each clinic and consisted of monitoring blood pressure and cholesterol and any medications taken by the patients, addressing any other patient health concerns, and performing routine health screenings. UCG participants also received incentives during the baseline and follow-up assessments. BODY.METHODS.COMMUNITY HEALTH WORKERS: The CHWs hired to deliver the program were of the same communities as the study sites and were required to be bilingual (in Spanish and English) and bicultural (have a similar heritage to program participants), have direct experience with Spanish-speaking immigrant communities, and have basic computer skills. Of the 8 CHWs hired, 7 were younger than 30 years, 6 had at least 2 years of college, and 2 had obtained postgraduate degrees in Mexico. All were women. All were paid salaries by the clinics, which used funds from the CDPH through a CDC grant. All CHWs remained employed by the clinics during the intervention and follow-up. Before the study began, CHWs received an intensive 21⁄2 days of initial training on how to deliver the enhanced intervention; the training was repeated 3 months into the study (8). The training included information on how to administer study protocols, conduct nutrition and physical activity behavior-change counseling, and collect self-reported data. In addition, CHWs and study staff participated in monthly 1-hour teleconference meetings. Training was provided by the California WISEWOMAN program staff and other state program partners. BODY.METHODS.MEASUREMENTS: Assessments. We assessed readiness to change behavior and self-reported physical activity by using 2 separate questionnaires during individual appointments 1 week after a woman's enrollment. Women began the intervention 1 month after their baseline assessment. Follow-up assessment was scheduled by appointment between 9 and 14 months after a woman's baseline assessment, and the average time to follow-up was 12 months. Of the 1,093 women selected to participate, 868 (79%) completed both the baseline and follow-up assessments. We were unable to contact the remaining 225 women (119 women in the EIG and 106 women in the UCG). The EIG and UCG at baseline did not differ in medication use, blood pressure, cholesterol, obesity rates, or 10-year coronary artery disease risk (10). Readiness for behavior change. At baseline and follow-up, CHWs administered a questionnaire to all participants on readiness to change 4 behaviors: 1) engaging in physical activity "hard enough to increase your breathing for at least 30 minutes 4 times a week," which we defined as vigorous activity; 2) taking up new physical activities; 3) adding intensity to daily activities (ie, performing normal activities such as vacuuming or gardening); and 4) changing daily habits to increase activity (eg, taking stairs instead of elevator). Possible responses to each question were, "No, I haven't thought about being more physically active and have no plans to change"; "No, but I am thinking about doing this sometime in the next 6 months"; "No, but I plan to start doing this sometime in the next month"; "Yes, I do this now, but it has been less than 6 months since I started"; "Yes, I do this now and have been doing this for 6 months or longer." We recoded these 5 possible responses into 2 variables: precontemplation/contemplation/preparation and action/maintenance. The format and design of the questionnaire were based on the work of Marcus and Simkin (13) and physical activity stages-of-change questionnaires adapted for low-income women (14) and low-income Mexican women (15). The stages-of-change model has good predictive validity for physical activity in low-income Mexican women in community settings (15) and low-income African American women in clinical settings (16). Cronbach α for the Spanish version of the questionnaire used in our study was α = 0.65. Self-reported physical activity. We measured self-reported physical activity at baseline and follow-up by using 2 items on the 8-item Spanish version of the Physical Activity Assessment survey developed by UNC-Chapel Hill (5,9) as part of the North Carolina WISEWOMAN program. We asked participants about their participation in walking or running by using the question, "Do you walk or run (for fun, exercise, transportation)?" We asked about participation in exercise or sports by using the question, "Do you do either of these [sports or exercise]?" We considered walking or running moderate physical activity and exercise or sports vigorous physical activity. The categories of intensity were validated against accelerometry (17). The answer for each question was yes or no. The Spanish version was adapted and translated with the curriculum as detailed earlier. BODY.METHODS.ANALYSES: We used data only from participants who completed both the baseline and follow-up assessments. We analyzed both dichotomous variables (stage of change and self-reported physical activity) in separate intent-to-treat mixed models using SAS version 9.1.3 (SAS Institute Inc, Cary, North Carolina) to account for the correlation within subjects of responses across time (18). The associations are expressed as odds ratios (ORs) and 95% confidence intervals (CIs). We examined post-hoc differences for interaction effects using the McNemar test for repeated measures and the χ2 test for group differences. BODY.RESULTS.PARTICIPANT CHARACTERISTICS: The demographic characteristics of the EIG and UCG did not differ significantly at baseline (Table 1). Women who self-reported data for both assessments did not differ in education from women had only a baseline assessment; however, they were more likely to identify as Mexican or Mexican American than women who had only a baseline assessment. BODY.RESULTS.SELF-REPORTED READINESS FOR CHANGE: Women in the EIG and UCG did not differ significantly at baseline in self-reported readiness to change any of the 4 physical activity behaviors assessed (Table 2). The EIG was twice as likely to report being in the action/maintenance stage for vigorous physical activity at follow-up than at baseline; 47% of women in this group reported readiness to engage in vigorous physical activity at baseline, and 67% at follow-up. The UCG's readiness for vigorous physical activity did not change between baseline and follow-up; 52% of women in this group reported readiness at baseline, and 58% at follow-up. Both groups reported significant increases in readiness to take up new physical activity, perform daily habits more briskly, and incorporate physical activity into daily activity. The increases were greater for the EIG than for the UCG. BODY.RESULTS.SELF-REPORTED PHYSICAL ACTIVITY: The EIG and UCG did not differ significantly at baseline in either category of self-reported physical activity (Table 3). The EIG was approximately twice as likely to report engaging in moderate physical activity at follow-up as at baseline; 71% of women in this group reported moderate physical activity at baseline, and 84% at follow-up. The UCG did not change between baseline and follow-up; 75% of women in this group reported moderate physical activity at baseline, and 77% at follow-up. The EIG was approximately 3 times as likely to report engaging in vigorous physical activity at follow-up as at baseline; 13% of these women reported moderate activity at baseline, and 33% at follow-up. Women in the UC group showed no change (16%, baseline vs 17%, follow-up). BODY.DISCUSSION: Only women in the EIG self-reported significant increases in both moderate and vigorous physical activity. More women in both groups at follow-up than at baseline reported being in the action/maintenance stages of readiness for 3 of the 4 physical activity behaviors. The change was greater for the EIG than for the UCG. Only the women in the EIG changed their readiness to engage in vigorous physical activity. Our findings support other studies showing that an increase in a person's readiness to change through health education alone often does not lead to changes in behavior (19). Health education is only 1 element of a comprehensive strategy for changing physical activity behavior, which also includes social support (20), reliance on self to overcome barriers (21), and restructuring the physical environment (22). These were all elements of the California WISEWOMAN intervention. In addition, research supports the addition of CHWs to health promotion research teams (23). CHWs help with recruitment (24) and retention (25) and may be more effective interventionists than traditional research staff (23). Our findings on changes in self-reported moderate and vigorous physical activity are similar to the findings of 4 studies that found significant increases in physical activity among Latinas (26-29). Most interventions for promoting physical activity among Latinas have intensive components such as exercise classes and walking several times a week for 2 to 10 months (29,30), frequent staff contact and counseling (26), and small sample sizes. It would be difficult to implement such interventions in clinical settings that have limited resources or provide services to thousands of patients annually. Our minimal-contact intervention (2 assessments and 3 behavior-change counseling visits) is much better suited to a variety of clinical settings that serve large numbers of low-income Hispanic/Latino patients who are at high risk for CVD. Our study had several limitations. We did not objectively measure physical activity as other studies have done (7,17). People over-report self-reported physical activity relative to physical activity measured objectively by activity monitors (31). However, self-report still provides more detail than activity monitors on the activities in which participants engage; activity monitors do not provide a comprehensive understanding of physical activity behavior (32). Our self-reported data is supported by previously reported results of the California WISEWOMAN intervention: women in the EIG reduced their 10-year coronary heart disease risk relative to women in the UCG (10). Another limitation is that we did not standardize the information or treatment of women in the UCG. Some of these women may have received information and attended classes on healthy lifestyles during the intervention. All women in our intervention were enrolled in the California NBCCEDP, so all may have received healthy lifestyle information through this program or another community program. These exposures are not likely to have accounted for the effect of our intervention, however, because all participants were equally exposed. A third limitation is that follow-up data were collected by the same CHWs who delivered the intervention. Although CHWs were trained to be aware of and avoid bias when collecting follow-up data, our findings would have been strengthened had we had different follow-up data collectors. Despite these limitations, our study has a number of strengths. One was the large sample size. Many published WISEWOMAN intervention studies had only one-quarter to one-half the sample size of our study (5-9,9,19). In addition, our study was conducted with strong design characteristics, such as randomization and standardization of the intervention protocol. We used the CHW model of health promotion, which is designed for community clinics that serve large populations of immigrant, non-English–speaking, low-income patients. Several studies have demonstrated the effectiveness of the CHW model for promoting health behaviors in community settings (23-25); ours is one of the first to demonstrate their important contribution to health promotion in a clinical setting. The California WISEWOMAN program is ideal for health promotion in clinical settings that serve low-income immigrant communities at high risk for CVD because of its strong foundation in behavior-change theory, its commitment to the CHW model, and its emphasis on initiating more vigorous physical activity.

TITLE: A prospective randomised radiostereometric analysis trial of SmartSet HV and Palacos R bone cements in primary total hip arthroplasty ABSTRACT.BACKGROUND: Introduction of new bone cements into clinical practice should include radiostereometric studies. ABSTRACT.MATERIALS AND METHODS: A prospective randomised radiostereometric study was performed, comparing SmartSet HV and Palacos R acrylic bone cements (without antibiotics) using third-generation cementing techniques in primary total hip arthroplasty. Thirty-five patients (36 hips) undergoing Charnley total hip arthroplasty were randomised to receive either of the two cements and were followed with repeated clinical, radiographic and radiostereometric examinations over 24 months. Twenty-seven patients (28 hips) attended 2 years postoperatively. ABSTRACT.RESULTS: The mean distal translation observed was −0.15 mm for SmartSet HV and −0.16 mm for Palacos R. The mean rotation around the longitudinal axis was 0.9° for SmartSet HV and 1.2° for Palacos R. The Merle d'Aubigne Postel score was the maximum of 18 points for all patients in both groups. ABSTRACT.CONCLUSIONS: No statistically significant difference in stem fixation with use of SmartSet HV and Palacos R was found at 2-year follow-up. BODY.INTRODUCTION: Many factors influence the long-term performance of cemented total hip replacement (THR) such as patient characteristics, the prosthetic components, bone cements and surgical techniques [1]. Increased prosthetic migration has been correlated with early loosening [2, 3]. This was clearly demonstrated when using Boneloc cement (Biomet, Warsaw, Indiana). This low-viscosity cement was found to be catastrophic based on a randomised clinical study using radiostereometric analysis (RSA) [4] and 5 years of data from the Norwegian Arthroplasty Register [5]. The RSA technique enables calculation of the three-dimensional translational and rotational movements of the implant relative to the bone with high precision and accuracy [6] and has become the gold standard for clinical evaluation of new surgical techniques and implants [7]. RSA is part of the recommended stepwise introduction of new surgical techniques and implants [8]. Fixation of the Charnley stem with Palacos R cement was previously found to be a good combination [9]. Although this manufacturer (Schering Plough, Belgium) no longer produces Palacos R, this cement was well established within the cement market, with many years of clinical success, at the time of conducting this clinical study. The SmartSet HV (DePuy CMW, Blackpool, UK) is a high-viscosity bone cement, which is self-curing and composed of methyl methacrylate (monomer) and methyl methacrylate/methylacrylate copolymer (polymer) (PMMA). This cement guarantees, according to the manufacturer, a "buffer zone" of viscosity prior to and after implant insertion, achieved by using a combination of two methyl methacrylate-methyl acrylate copolymers. Such a buffer zone may contribute to more controlled pressurisation during implant insertion. Preclinical mechanical tests revealed no differences in mechanical properties between Palacos R and SmartSet GHV (SmartSet HV with gentamicin added) when tested at 20°C, whereas no comparisons at 37°C were made [10]. The objective of this prospective randomised study was to investigate early migration of the Charnley femoral prosthesis when implanted with either SmartSet HV or Palacos R bone cement. Neither of the cements contained an antibiotic. BODY.MATERIALS AND METHODS: The study was conducted in accordance with the Declaration of Helsinki and approved by the Norwegian Technical and Scientific University Central Region Ethics Committee (reference 094-02). The patients included were recruited from patients consecutively admitted to a single study centre (Orthopaedic Department, St. Olavs Hospital, Trondheim University Hospital Norway). Excluded from participation were any patients with an existing condition such as malignancy, pregnancy, severe osteoporosis and disabling musculoskeletal problems (other than in the hips), patients on corticosteroid treatment and patients who had already participated in a clinical study with an investigational product in the last 6 months. Thirty-five patients were asked to participate, and all agreed. All patients signed an informed consent. Thirty-six hips (35 patients) were included (Table 1).Table 1Clinical characteristics of each groupSmartSet HVPalacos RHips included (n)18Male (4), female (14)18Male (8), female (10)Diagnosis (n)16 Primary osteoarthritis1 Congenital hip dysplasia1 Post-traumatic arthritis17 Primary osteoarthritis1 Avascular necrosisAge at surgery, years (n = 36)Mean (range)69 (57–78)69 (59–77)Weight (include patients), kgMean (range)75 (60–90)78 (52–98)Lost to follow-up (n)4Missing baseline RSA examination (2)Death of patient before 2-year follow-up (2)4Missing baseline RSA examination (2)Did not attend arranged RSA examinations (2)Returning for follow-up (n)3 months (n = 14)6 months (n = 10)1 year (n = 16)3 months (n = 13)6 months (n = 10)1 year (n = 16)Hips attending at 2-year follow-up (n)14Male (2), female (12)14Male (5), female (9)Diagnosis at 2-year follow-up (n)13 Primary osteoarthritis1 Congenital hip dysplasia13 Primary osteoarthritis1 Avascular necrosisAge at surgery, years (n = 28)(2-year follow-up population)Mean (range)69 (62–77)70 (59–74)Weight at surgery, kg (n = 28)(2-year follow-up population)Mean (range)74 (60–85)76 (52–90) The Merle d'Aubigne Postel score [11] was recorded preoperatively as a baseline clinical evaluation of the patient's level of pain, mobility and walking ability. Absence of pain, mobility of greater than 90° flexion and 30° abduction, and normal, unlimited walking ability gives a maximum score of 18 points. The cement randomization was electronically generated preoperatively (SAS version 8), with equal numbers of patients randomly allocated to each of the two cements. No stratification was used. Each code was kept in a sealed opaque envelope and broken during the operation, immediately before cementing. The single patient with bilateral hips included was randomised to receive the Palacos R cement in both. One experienced surgeon (O.S.H.) performed the surgical intervention on all 36 hips, from October 2002 to October 2003. The surgeon was not blinded in respect to the cement used. This was impossible due to the differences in colour and handling of the two types of cements. The patients were blinded to which cement they received. The RSA technique employed requires tantalum beads fixed on the implant and beads implanted into the bone. All patients received a Charnley flanged 40 prosthesis (DePuy International, Leeds UK), manufactured with tantalum bead mounted stainless-steel towers; one tower was fixed to the distal tip of the stem and one in the proximal shoulder region. A third marker is referenced by the central point of the Charnley 22.225-mm-diameter femoral head. A posterolateral surgical approach (the hospital standard) was used, with a lateral incision [12]. A neck resection guide was used, and the femoral canal was entered through the piriformis fossa. The femur was prepared with a 12-mm central reamer, followed by a 1-mm all-direction oversized broach, and rinsed with saline using pulsed lavage. Bleeding was controlled at the prepared surfaces with use of a 1% adrenalin-soaked sponge. Nine tantalum beads (0.8 mm in diameter) were implanted into the bone inside the prepared femoral cavity, ideally five in the greater trochanter and four in the lesser trochanter region. Third-generation cementing technique incorporating retrograde filling and proximal occlusion during pressurisation was used for all patients. Distal cement restriction was obtained using a polyethylene plug (Cement Restrictor, DePuy International, Leeds, UK). Palacos R was prechilled for minimum 24 h to 8°C before mixing. SmartSet HV was stored at 21°C. Both cements were mixed under vacuum, using the Cemvac system (DePuy CMW, Blackpool UK) and a syringe and gun. All patients received the standard Charnley Ogee cup (DePuy International, Leeds, UK). The same cement type was used for both the femoral and acetabular component. Standard radiographic examination (AP and lateral view) of the hips was carried out. Implant position relative to the central axis of the femur was described by manually measuring the alignment of the central axis of the stem with respect to the axis of the femoral canal. Discrepancies of greater than ±5° from the neutral position would be classified as malalignment. The baseline RSA radiographic examinations were carried out as soon as the patients were capable of weight bearing and walking after surgery, typically within 7 days. RSA evaluation was performed by using UmRSA software (version 6.0, RSA Biomedical Innovation, Umea, Sweden). The mean error fitting upper limit was set to 0.25 mm, and the upper limit for the condition number 150. The coordinate axes and directions of the rotations are illustrated in Fig. 1.Fig. 1The Charnley stem, labelled with the axes and directions of the rotations used. Tantalum markers embedded in the bone and mounted on the femoral stem are visible RSA operator was not blinded to the cement type used. We believe this to be acceptable. The RSA software automatically identifies markers and thereby reduces possible operator-induced biases. The patients returned for postoperative follow-up at 3, 6, 12 and 24 months. At each follow-up, standard radiographs, RSA and completion of the Merle d'Aubigne Postel score were recorded. Only ten hips in each group attended at the 6-month RSA follow-up, and these RSA measurements were therefore excluded from publication due to the low number of participants. BODY.STATISTICAL METHODS: The sample size calculation was based on micromotion data of Charnley stems in combination with Palacos R cement earlier presented from our research laboratory [13] using Sample Power 2.0 (SPSS Inc., Chicago, USA). With sample size of 18 in each group, α = 0.05 and β = 0.80, an alternative mean of more than ±0.06 mm of migration and more than ±0.92° of rotation (translation along and rotations around the Y-axis) could be detected. An assumption of equal variance in both groups was made. Testing of normality of distribution was performed with Q–Q plots. Two-tailed independent t-test was used to analyse differences in micromotion between the two cement groups. A probability level of P < 0.05 indicated statistical significance. The precision of the RSA technique was measured by double examinations of 22 patients at their 2-year follow-up visit. Each patient got up from the X-ray table and walked in the room between the two examinations, which were made within 10 min of each other. The precision values were calculated as follows: first, the differences between the two examinations for each patient were calculated. Second, the standard deviation (SD) of these differences with respect to zero (not to the mean) was calculated [14].\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\text{SD}} = \sqrt {{\frac{{\sum\nolimits_{i = 1}^{n} {\left( {x_{i} } \right)^{2} } }}{n}}}, $$\end{document}X represents the differences between the double examinations (n = 22). Finally, the SD multiplied by 2.074 (representing the 0.975 percentage point at a t22 distribution) defines the precision. BODY.RESULTS: The micromotion measurement results are presented in Fig. 2 and further in Table 2.Fig. 2(a) Rotation around the Y-axis. The graphs present mean and standard error of the mean. (b) Translation along the Y-axis. The graphs present mean and standard error of the meanTable 2Observed micromotion at 2-year follow-up, mean (SD)AxesRotation (°) (SD)Translation (mm) (SD)XYZXYZSmartSet HV−0.13 (0.49)0.91 (0.71)−0.08 (0.18)0.08 (0.11)−0.15 (0.08)−0.06 (0.22)Palacos R−0.21 (0.35)1.19 (0.96)−0.16 (0.16)0.03 (0.08)−0.16 (0.06)0.02 (0.23)Difference0.08−0.280.080.050.01−0.0895% CI of the difference Upper0.420.370.210.130.070.10 Lower−0.25−0.94−0.05−0.02−0.05−0.25 P value0.600.380.200.160.740.36CI, confidence interval The error fitting limits and conditions were met in all RSA analyses, and the measurement data were found to be normally distributed for all six data sets. At 2-year follow-up there were no statistically significant differences in rotations or translations between the SmartSet HV group and the Palacos R group. The precision data are presented in Table 3.Table 3Precision of RSAAxesRotationTranslationXYZXYZMean difference of first and second reading0.050.070.010.000.01−0.012.074 × SD0.431.010.120.100.100.22Precision values are based on 22 double examinations at 2-year follow-up The Merle d'Aubigne Postel score was completed for each patient preoperatively and at 2-year follow-up. Preoperatively, the mean score and range was 10 (7–13) for the SmartSet HV group and 10 (7–12) for the Palacos R group. At 2 years, the score was the maximum of 18 points for all patients in both groups. AP and lateral X-ray analysis indicated that all implants were positioned neutrally relative to the central axis of the femur. All cement mantles in both groups were intact and complete white-out in the bone interface was registered on all postoperative radiographs. A total of three severe adverse events have been reported, none likely to be study related. One patient suffered from cerebral infarction, which occurred in the immediate postoperative period. This patient recovered fully and continues to be followed up in this investigation. Two patients died: one due to multi-organ failure, the other as a result of pneumonia. None of these patients had undergone a hip revision. BODY.DISCUSSION: Introduction of new orthopaedic products into clinical use should be done with great care. Kärrholm et al. [15] recommend investigation in small trials, before entering larger clinical trials, when introducing new orthopaedic products. The aim is to limit possible hazards to as few patients as possible. Thanner et al. evaluated the properties of Boneloc® and included 30 patients, of whom 14 received Boneloc®. The SmartSet GHV cement was included in an in vitro study of five different PMMA bone cements [16]. Differences in viscosity behaviour, and waiting and hardening time were found between the cements investigated, leading to the conclusion of the necessity of RSA studies before any broad clinical use of new cements. The RSA method is highly accurate and precise and therefore well suited when small study populations are involved [17]. However, small clinical trials are susceptible to biased patient selection and loss to follow-up, both experienced in the present study. We found an almost unacceptable loss to follow-up, and retrospectively more patients should preferably have been enrolled. Eighteen hips were initially included in each group; however, data only from 14 hips in each group were available at 2-year follow-up. Consequently there was loss of statistical power. With use of the results presented in Table 2 and Sample Power 2.0, these losses could be quantified. Differences in subsidence greater than or equal to 0.09 mm and/or rotation greater than or equal to 1.06° could be described as statistically significant (translation along and rotations around the Y axis, α = 0.05 and β = 0.80, independent t-test). The loss of statistical power seemed to be acceptable. The patient group at 2-year follow-up was imbalanced in gender distribution. However, we believe the gender distribution not to impair the results presented. A large clinical study (n = 3,461) assessing differences between gender in clinical outcome found no such inequalities [18]. Olofsson, Önsten and Kärrholm reported no influence of patient factors such as gender on stem fixation [9, 19, 20]. A logistic regression analysis based on patient factors, type of operation and RSA data revealed that the amount of subsidence after 2 years is the best predictor of revision [2]. The distal migration of the Lubinus SP I femoral stem at 2 years predicted stem survival. Subsidence greater than 1.2 mm was associated with more than 50% stem revision [2]. Correlations between short-term prosthetic micromotion and future risk of prosthetic failure have been demonstrated in studies involving both hip and knee prostheses. Kärrholm et al. found it difficult to define acceptable limits of short-term prosthetic micromotion [7], while Ryd et al. [21] set the limit of distal migration at 0.2 mm. Micromotions of the Charnley flange 40 stem combined with Palamed G and Palacos R with gentamicin are described by Hallan et al. [13]. At 2-year follow-up, internal rotation of 1.7° and 2.0° was found. The subsidence was 0.18 and 0.21 mm, respectively. Grant et al. [22] found internal rotation of 1.1° at 2-year follow-up using Charnley Elite Plus in combination with Palacos R with gentamicin. Subsidence was less than 0.18 mm. The levels of micromotion found in the present 2-year RSA study were similar to those presented by Grant and Hallan. There were no significant differences in either translation or rotation between SmartSet HV and Palacos R bone cements, the latter having documented good long-term clinical results [17, 23, 24]. For both cements, mean stem subsidence was smaller than 0.2 mm, which is considered to be satisfactory with respect to long-term performance [7, 9, 22]. One hip in the Palacos R group rotated much more than the others: 3.67° at 2 years. The subsidence for this hip at 2 years was 0.19 mm. Two hips in the SmartSet HV group had subsidence of 0.32 mm at 2 years. Data from these three hips are included in the study. Other studies have excluded such cases [13]. The internal rotation was, as expected, higher the first year than the second. Though there was no statistically significant difference in internal rotation, it seemed to be slightly smaller in the SmartSet HV group. Our baseline RSA examinations were obtained, as recommended in Guidelines for standardisation of radiostereometry, before the patients had started the more active part of mobilisation [17]. As a consequence, we believe the results presented not to be underestimated. Those guidelines also advise double examinations to verify the precision of RSA data and that all research groups employing RSA should obtain and present their precision data. A precision of 1.0 degrees for internal rotation and 0.1 mm for subsidence was found in the present study. The calculation method is described in detail, since several methods have been employed for calculating such data [13, 14, 22, 25]. This makes comparison of our precision data with other publications somewhat difficult. However, we believe the method presented herein to be best suited and our precision data to be good. The present study was dimensioned to measure movements between the implant with respect to the bone only. No attempt was made to separate possible movements between the implant and the cement mantle from movements between the cement mantle and the bone. For this reason no tantalum beads were embedded in the cement nor implanted into the cement restrictor. Sundberg et al. and Hallan et al. deployed tantalum markers both in the bone and in the cement [13, 25]. Both studies concluded that micromovement occurred between the femoral component and the cement. The cement mantle was stable with respect to the bone. Stefánsdóttir et al. [26] described some small movement of the cement mantle, whereas movements mainly occurred inside the mantle. Technical obstacles in employing the RSA technique to describe cement mantle movement were experienced in these studies. The tantalum markers embedded into the cement could be both poorly spatially distributed and difficult to visualise. As a consequence, a high number of patients had to be excluded when describing movement of the cement mantle. Long-term follow-up data from clinical trials and national registry reports are needed to ensure safe practice in joint replacement. RSA studies can help to predict the long-term results when levels of micromotion are concerned. In conclusion, the Charnley flange 40 stem in combination with the two bone cements investigated performed well at 2-year follow-up.

TITLE: A randomized 3x3 crossover study to evaluate the effect of Hass avocado intake on post-ingestive satiety, glucose and insulin levels, and subsequent energy intake in overweight adults ABSTRACT.BACKGROUND: The behavioral outcome of food ingestion is a complex process that involves psychological and biological factors. Avocados are nutrient dense with properties that may favorably impact energy balance. This study sought to evaluate if incorporating approximately one half of a Hass avocado by addition or inclusion into a lunch meal will influence post-ingestive satiety, glucose and insulin response, and subsequent energy intake among overweight adults. ABSTRACT.METHODS: This was a randomized 3x3 single-blind crossover design study with 26 healthy overweight adults (mean ±SD age 40.8±11.0 years and BMI 28.1±2.4 kg/m2). Participants consumed a standardized breakfast followed by 1 of 3 lunch test meals [Control (C), avocado-free; Avocado Inclusive (AI); and, Avocado Added (AA)]. Participants rated five appetite sensations using a visual analog scale (VAS) before lunch and at specific intervals over 5 hours following the start of the test meal. Blood glucose and insulin were measured before lunch and at specific intervals over 3 hours following the start of the test meal. Mixed models were used to compare differences among the 3 test meals, and the area under the curve (AUC0-xh) was computed for the VAS and biological measures. ABSTRACT.RESULTS: There were significant differences in the AUC(0-5h) for the self-reported feelings of satisfaction (P=0.04) and desire to eat (P=0.05) in the mixed model analysis. Compared to the C test meal, the AA test meal increased satisfaction by 23% (P=0.05) and decreased the desire to eat by 28% (P=0.04) for the AUC(0-5h). For the AUC(0-3h), the AA test meal increased satisfaction by 26% (P=0.02) and decreased the desire to eat by 40% (P=0.01) as compared to the C test meal. Compared to the AI meal, the AUC(0-3h) for blood insulin was higher in the C and AA meals (P=0.04 and P=0.05, respectively). ABSTRACT.CONCLUSIONS: The addition of approximately one half of a Hass avocado at a lunch meal can influence post-ingestive satiety over a subsequent 3 and 5 hour period in overweight adults. A caveat to these findings is that the avocado contained an additional 112 kcal, which may have accounted for the observed increase in satisfaction and decreased desire to eat. Future trials are warranted to evaluate the effects of avocado intake on weight management in adults of varying BMIs and among insulin resistant individuals. BODY.BACKGROUND: The behavioral outcome of food ingestion is a complex process that involves psychological and biological factors that culminates in an individual's overall 24-hour energy intake [1]. One of the components of the appetite system is satiety, which reflects a process that leads to increased fullness after a meal, a decline in hunger, and inhibition of further eating in the postprandial period. In addition to sensory quality, the macronutrient composition, physical structure and energy density of a whole food may contribute to the modulation of satiety. More recent research has shown that the volume of a meal also influences satiety [2,3]. Additionally, the quality of the fat composition, i.e. degree of saturation of fatty acids in a food, may influence rates of oxidation and thermogenesis in animals and humans [4,5]. Furthermore, a single food may favorably impact energy balance according to its ability to offset spontaneous energy reduction at the next meal(s), which is known as the dietary compensation score [6]. The fresh pulp weight of Hass avocados is 72% water and contains only 1.7 kcal/g, therefore they are classified as a medium energy dense food (defined as a range between 1.5-4.0 kcal/g) [7]. Hence, when added to a meal they will increase the volume similar to other fruits and vegetables, which are food categories that have been previously shown to have a beneficial effect on weight control [8]. Further, the typical serving size is approximately one half of a medium size avocado (70 g) [9], which translates to being an excellent source of dietary fiber (5 g). Fiber is another food component strongly linked to enhancement of satiety [10] and modulation of the glucose and insulin responses to meals [11]. The connection between potential biological markers of appetite regulation continues to be an active area of research in normal weight and overweight populations. A 2007 meta-analysis by Flint et al. [12] has shown that the biological signaling of satiety by insulin in the overweight population is blunted, which could lead to the deleterious consequence of overeating at subsequent ad libitum meals and snacks. In light of the increased prevalence in overweight in humans and cross-sectional evidence showing an increase in snacking and total energy intake in the United States over the past three decades [13], the addition of approximately one half of an avocado at a specific meal(s) may be a simple dietary intervention to consider for individuals that consume excessive energy during specific snack and/or meal times. The aims of the present study are to evaluate if incorporating ~1/2 of a fresh Hass avocado by addition or inclusion into a lunch meal will influence post-ingestive satiety, the glycemic and insulin response, and subsequent energy intake in overweight adults. BODY.METHODS: We conducted a randomized 3x3 single-blind crossover design study (three 1-day study periods scheduled 1 week apart) at Loma Linda University, Loma Linda, California. Using a within subject repeated measures design, we evaluated the effect of avocado intake on the short-term regulation of food by employing the use of one of three lunch test meals within a single day on three different days. Each participant received all treatments on the same day of the week and had a 1 week washout period between treatments. BODY.METHODS.ELIGIBILITY CRITERIA: Healthy overweight and moderately obese adults were recruited through the use of posters, flyers, and newspaper advertisements on the Loma Linda University campus and in the surrounding communities. A study web page was developed with a complete description of the study and online application form. Applications were also taken by phone. Eligibility criteria were: age 25–65 years, body mass index (BMI, kg/m2) ≥25 and ≤35, weight stable for at least 6 months, normoglycemic, normotensive, sedentary or low level of habitual activity (less than 10 hours of exercise per week), non-smoker, not dependent on caffeine, and not taking any medications known to influence postprandial glucose and insulin levels. The recruitment process yielded 80 applicants and 56 individuals met the eligibility criteria. Forty-seven individuals attended information meetings and 30 were selected, plus 2 alternates. Four of the selected applicants declined participation due to unforeseen scheduling conflicts. Both alternates were included to achieve the targeted accrual of 28 participants and goal of 25 completers for adequate power (see Statistical Methods). One participant withdrew on the first day and one participant was asked to leave the second week due to non-compliance with the study protocol. The study was approved by the Loma Linda University Institutional Review Board and informed written consent was obtained from all participants. BODY.METHODS.ANTHROPOMETRIC MEASUREMENTS: Height was measured to the nearest centimeter using a stadiometer on the first study day. Weight was measured using an internally calibrated segmental body composition scale/analyzer (model TBF-300A, Tanita®, Arlington Heights, IL) and recorded to the nearest 0.01 pound. BMI was calculated as weight(kg)/height(m2). The daily energy needs for each participant were estimated using the Harris-Benedict equation after adjustment for overweight status, which was subsequently multiplied by an activity factor of 1.3 for sedentary lifestyle. Participants were then assigned to receive either a 1600, 2000 or 2400 kcal daily meal plan. BODY.METHODS.STUDY MEALS: Under the direct supervision of trained study personnel in the Loma Linda University Department of Nutrition Metabolic Kitchen, participants consumed the same standardized breakfast meal containing 25% of their estimated daily energy needs on each of the 3 assigned study days. For lunch, participants consumed 1 of 3 test meals: Control (C), avocado-free; Avocado Inclusive (AI); or, Avocado Added (AA) (see below). The standardized breakfast and the lunch test meals were designed to deliver the recommended levels of macronutrients according to the Acceptable Macronutrient Distribution Ranges developed by the Food and Nutrition Board of the Institute of Medicine [14]. The participants consumed 13-14% energy from protein, 49-51% energy from carbohydrate, and 35-38% energy from fat at the three lunch test meals. Further, the C and AI lunch meals delivered 35% of the participant's daily energy needs and the AA lunch meal provided 41% of the daily energy needs (Table 1). All foods were precisely measured or weighed to the nearest gram using a digital scale and the meals were matched for taste and appearance. Participants were permitted to drink water with and between meals on the three study days according to their typical pattern of water intake. Table 1 Percent of daily energy and macronutrient content of the 3 lunch test meals   Control Avocado Inclusive Avocado Added Energy, % 35 35 41 Carbohydrate, % 51 50 49 Protein, % 14 14 13 Fat, % 35 36 38 The standardized breakfast meal included orange juice, cornflakes, milk and a commercially prepared scone. The C test meal included a salad (mixed greens, cherry tomatoes, reduced fat Swiss cheese, Italian salad dressing), a refined grain French baguette and commercial chocolate chip cookies. Fresh, ripe Hass avocados (provided by the Hass Avocado Board) were sliced and included or added to the C test meal to produce the AI and AA test meals, respectively. The amount of avocado varied (range of ~50 to 90 g) with the energy needs of the participant [75 g (~1/2 of an avocado) for the 2000 kcal meal plan]. The portion sizes of the salad dressing and cookies were reduced in the AI test meal to match the energy and macronutrient content of the C test meal (Table 1). The dinner buffet meal on the 3 study days was served 5 hours from the start of the lunch test meal and contained a variety of foods with pre-identified portion weight, macronutrient and calorie content. Participants were allowed to consume sweet and savory food options that differed in energy density in an ad libitum manner to allow for the assessment of postprandial food intake and dietary compensation. The number of portions of food items selected and consumed by the participants was directly observed and written in a discreet manner by a senior investigator and trained research staff. To supplement the written documentation of the food items taken and leftovers remaining on the plate, a hidden video camera was utilized to record the foodservice delivery process. The leftovers were photographed using a digital camera and weighed to the nearest gram using a digital scale. Two research assistants separately compared the written documentation with the videotape recording, still photos and weight of leftovers to produce a record of food intake for each participant. If any discrepancy existed between the two researcher's records, a senior investigator reviewed all of the data sources to determine the most valid measurement of dietary intake. Pre-portioned evening snacks were provided to participants at the conclusion of the ad libitum dinner meal and participants were asked to record any snacks consumed after leaving the research kitchen until going to bed or until midnight. Participants were contacted by phone the following morning by study personnel for a self-report of the intake of pre-portioned evening snacks. The energy and macronutrient intake subsequent to the lunch test meals was assessed based on the observed food consumed at the ad libitum dinner meal and from the participant's self-reported consumption of pre-portioned evening snacks. BODY.METHODS.VISUAL ANALOG SCALES: By means of a mark on 100 mm line visual analog scales (VAS), participants rated their appetite sensations (hunger, fullness, satisfied, desire for a meal, and prospective food consumption). The VAS was completed before lunch and at approximately 30, 60, 90, 120, 180 and 300 minutes following the lunch test meal on each study day. The five scales were anchored at the low end with the most negative feelings (e.g. not at all) and opposing terms at the high end (e.g. extremely high). BODY.METHODS.SAMPLE COLLECTION AND LABORATORY ASSESSMENT: On the 3 assigned study days, participants arrived in the morning to the Nutrition Research Laboratory (NRL) after a 12-hour overnight fast for a baseline blood draw to measure glucose and insulin concentrations. Participants were free to engage in their normal morning routines but were instructed to return to the NRL by noon. The lunch test meal (C, AI or AA) was ingested within a 30 minute time period and additional blood samples were taken at approximately 30, 60, 90, 120 and 180 minutes following the start of the lunch test meal. Venous blood samples were drawn and collected into vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) and centrifuged at 1500 x g at 4°C for 10 min. Serum and plasma were separated, aliquoted and frozen at −80°C until analyzed. Serum glucose was assayed with the glucose-oxidase-peroxidase enzymatic assay using kits supplied by Cayman Chemical (Ann Arbor, MI). Serum insulin was assayed using ELISA kits supplied by ALPCO Diagnostics (Salem, NH). BODY.METHODS.STATISTICAL METHODS: Sample size, power calculations and statistical analysis were performed utilizing SAS version 9.3 (SAS Institute, Cary, NC). All tests were two-sided and a value of P < 0.05 was considered significant. Under good experimental laboratory conditions, a sample size of 20 to 25 participants has been shown to be adequate to denote a 10% difference in AUC appetite ratings, which is considered to be a reasonable difference [15]. The target accrual was 28 participants to allow for a 10% dropout rate, which has been the observed dropout rate for our prior short-term feeding studies. A mixed model statistical approach was used to compare differences among the 3 test meals adjusting for study periods as fixed effects and treating participants as random effects. When significant findings were observed, Tukey post-hoc testing was performed to further elucidate the differences between the 3 test meals. The weighted mean dietary intake of avocado was computed based on the number of participants assigned to each of the three aforementioned energy levels. The dietary compensation score across the C and AA test meals was calculated at the individual level using the following equation [6]: Percent Dietary Compensation = (Intake without load, C)-(Intake with load, AA)/Energy content of load x 100. More specifically, the dietary compensation at dinner was computed at the individual level by subtracting a subject's dinner intake after the C lunch test meal minus the same subject's dinner intake on the day of the AA lunch test meal, divided by the energy (or macronutrient) from the avocado consumed. Two trained research assistants measured the VAS data to the nearest 0.1 cm and any discrepancy was resolved by a senior investigator. To compute the area under the curve (AUC) from zero to x hours (AUC(0-xh)), the minimum value of each subjectively reported VAS scale (in mm) over time was determined at the individual level and then the AUC above the minimum value was calculated using the linear trapezoidal rule. The AUC is reported as mm x minutes and was constructed by plotting the subjective values between 0 to 100 mm over time (minute) for each of the five VAS questions. The VAS was completed and blood samples were scheduled to be taken at approximately 30, 60, 90, 120, and 180 minutes following the test meal. The exact time for each individual VAS and blood sample collection was recorded and these times were used for the analysis. Curved lines were generated to show the area under the curve for glucose and insulin (Figure 1) and the five VAS questions (Figure 2), which better represent the reality of the study data collection and analysis. Figure 1Blood glucose and insulin levels after consumption of the 3 lunch test meals. Three-hour area under the curve AUC(0-3h) based on difference from baseline (time 0) is shown as an insert. Compared to the AI test meal, the blood insulin was higher in the C and AA test meals (P = 0.04 and P = 0.05, respectively). Figure 2Ratings for the five visual analog scale questions after consumption of the 3 lunch test meals. Five-hour area under the curve AUC(0-5h) based on difference from baseline (time 0) is shown as an insert. Compared to the C test meal, the AA test meal increased satisfaction by 23% (P = 0.05) and decreased the desire to eat by 28% (P = 0.04). Data are expressed as adjusted mean ± SE unless otherwise noted. BODY.RESULTS: The 26 participants that completed the study consisted of 16 women and 10 men with a mean ± SD age 40.8 ± 11.0 years and BMI 28.1 ± 2.4 kg/m2. Ten participants consumed the 1600 kcal meal plan, nine consumed the 2000 kcal meal plan and seven consumed the 2400 kcal meal plan. The weighted mean dietary intake of avocado was 67.5 grams, which contained 112 kcal, 1.3 g protein, 5.6 g carbohydrate and 10.4 g fat. BODY.RESULTS.BLOOD GLUCOSE AND INSULIN CHANGES: There were no significant differences between the 3 lunch test meals for AUC(0-3h) blood glucose (Figure 1). Compared to the AI test meal, the AUC(0-3h) for blood insulin was higher in the C and AA test meals (P = 0.04 and P = 0.05, respectively). Difference in blood insulin levels between treatments were observed at the 30 minute time point (P = 0.04) as follows: adjusted mean (95% CI) for C = 54μIU/ml (39, 74); AI = 34μIU/ml (25, 47); and, AA 42 μIU/ml (30, 57). BODY.RESULTS.VISUAL ANALOG SCALE CHANGES: There were significant differences in the AUC(0-5h) for the self-reported subjective feelings of satisfaction (P = 0.04) and desire to eat (P = 0.05) in the mixed model analysis (Figure 2). Post-hoc testing revealed that compared to the C test meal, the AA test meal increased satisfaction by 23% (P = 0.05) and decreased the desire to eat by 28% (P = 0.04) for the AUC(0-5h). For the AUC(0-3h), the AA test meal increased satisfaction by 26% (P = 0.02) and decreased the desire to eat by 40% (P = 0.01) as compared to the C test meal (Table 2). Additionally, the AI test meal showed a tendency towards increasing satisfaction by 22% P = 0.07) as compared to the C test meal for the AUC(0-3h). Lastly, the five measurements of appetite sensation tended to converge 5 hours after the lunch test meal. Table 2 Three-hour area under the curve AUC a (0–3h) based on difference from baseline (time 0) for the five visual analog scale questions between the 3 lunch test meals   Control Avocado Inclusive Avocado Added   Question Mean b SE c Mean b Difference d P-value e Mean b Difference d P-value e P-value f How hungry are you? 3105 394 2358 −24% 0.30 2418 −22% 0.36 0.26 How strong is your feeling of fullness ? 7249 771 8107 +11% 0.64 8189 +11% 0.58 0.55 How satisfied are you? 6340 726 8149 +22% 0.07 8562 +26% 0.02 0.02 How strong is your desire to eat? 2993 319 2263 −24% 0.16 1798 −40% 0.01 0.01 How much do you think you can eat? 2641 318 2185 −17% 0.45 2031 −23% 0.24 0.25 a The AUC is reported as mm x minutes and was constructed by plotting the subjective values between 0 to 100 mm over time (minute) for each of the five VAS questions. b Adjusted mean from the mixed model analysis. c Common standard error (SE) for all of the adjusted means. d Percent difference compared to Control lunch test meal. e P-value compared to Control lunch test meal. f P-value for diet effect from the mixed model analysis. BODY.RESULTS.INTAKE AT THE AD LIBITUM DINNER MEAL AND EVENING SNACK: Dietary intake at the subsequent ad libitum dinner meal and evening snack after each of the 3 lunch test meals was equivalent for total energy, macronutrients and percent energy from the macronutrients (Table 3). Compared to the C test meal, the percent dietary compensation for the AA test meal for energy, protein, carbohydrate and fat was 66%, 235%, 118% and 36%, respectively. Table 3 Intake from the dinner meal and evening snack after the 3 lunch test meals   Control (C) Avocado Inclusive (AI) Avocado Added (AA) P-value c % Dietary Compensation d   Mean a SE b Mean a Mean a   Mean SE Energy (kcal) 1276 82 1193 1194 0.37 66 64 Protein, g (PRO) 42.6 2.8 39.2 39.0 0.27 235 204 Carbohydrate, g (CHO) 134.6 8.6 128.6 126.1 0.37 118 134 Fat, g 64.2 4.6 59.3 60.7 0.47 36 37 PRO, % total energy 13.4 0.3 13.2 13.0 0.36 -   CHO, % total energy 42.8 1.1 43.5 42.6 0.72 -   Fat, % total energy 44.7 1.1 44.3 45.4 0.58 -   a Adjusted mean based on the mixed model analysis. b Common standard error (SE) for all of the adjusted means. c P-value for diet effect from the mixed model analysis. d % Dietary Compensation = Intake without load , C – Intake with load , AA × 100 Energy content of load . [Note: The % dietary compensation at dinner was computed at the individual level by subtracting a subject’s dinner intake after the C lunch test meal minus the same subject’s dinner intake on the day of the AA lunch test meal, divided by the energy (or macronutrient) from the avocado consumed]. BODY.DISCUSSION: The results of this study suggest that the addition of ~ 1⁄2 of a Hass avocado at a lunch meal can influence post-ingestive satiety over a subsequent 3 hour and 5 hour period in overweight and moderately obese adults. Specifically, adding avocado to a lunch meal yielded a 23% increase in satisfaction (P = 0.05) and a 28% decreased desire to eat (P = 0.04) over a subsequent 5 hour period as compared to the avocado-free control lunch meal. Also, adding avocado to a lunch meal yielded a 26% increase in satisfaction (P = 0.02) and 40% decreased desire to eat (P = 0.01) as compared to the avocado-free control lunch meal over a 3 hour period. However, an additional 112 kcal was contained in the avocado, which may have accounted for the observed increased satisfaction and decreased desire to eat. Further, a 24% decreased desire to eat (P = 0.16) and 22% increase in satisfaction (P = 0.07) was observed over a 3 hour period after consumption of the isocaloric avocado inclusive lunch test meal as compared to the avocado-free control lunch meal. However, the changes in all five measurements of appetite sensation tended to taper off after 5 hours. Energy intake at the subsequent ad libitum dinner meal and evening snack and dietary compensation did not differ between the 3 lunch test meals, which may have been due to the 5 hour time interval between the lunch test meal and ad libitum dinner meal. De Graaf and Hulshof [16] have previously reported that the weight or amount of food in a preload affects subsequent appetite and food intake for only up to two hours after the preload. These findings are consistent with the findings of equivalent energy intake at the subsequent dinner meal and evening snack in the current study, yet inconsistent with changes in two specific measures of appetite sensation that we observed at both 3 and 5 hours for the avocado added test meal. Further, Flint et al. [15] has reported that an 8-10% difference in the response magnitude relative to control in food intake or satiety score (AUC) is of practical relevance. We found differences of practical relevance for all five appetite sensation measurements between the C versus the AI and AA interventions ranging between 11-24% and 11-40%, respectively. However, we did not find a statistically significant difference for hunger, fullness or prospective food consumption between the 3 test meals. Our overweight participants partially compensated for energy (66%) and fat (36%) intake and overcompensated for protein (235%) and carbohydrate (118%) at a subsequent ad libitum dinner meal and evening snack when avocado (weighted mean energy = 112 kcal) was added to the lunch meal. Thus, the majority of the energy provided by the addition of avocado to the diet was offset by dietary adjustments at the ad libitum dinner meal and evening snack. Others have reported that individual daily energy intake can vary by 20 to 30 percent, and that short-term dietary manipulations of less than ~400 kcal may not heavily influence dietary energy compensation [17,18], which may have been one of the reasons for the equivalent subsequent energy intake at the dinner meal and evening snack between the 3 study days. There are two potential ways a whole food can be incorporated into a meal, addition or isocaloric replacement. Addition is when the food is simply added to a meal, which results in an increase in nutrients and total energy, whereas isocaloric replacement occurs when the food is included and other foods are simultaneously decreased or eliminated to compensate for the overall energy content of the meal. It is worth noting that the AUC(0-3h) for blood glucose in the current study was equivalent between the 3 lunch test meals despite the additional mean energy (112 kcal) content and additional ~7 g carbohydrate in the AA lunch test meal. Avocados contain a unique seven carbon sugar (D-manno-heptulose) that does not contribute energy, and some believe it may support blood glucose control and weight management by reducing glycolysis via hexokinase inhibition [19]. Additionally, 30 minutes after the start of the lunch test meal the inclusion and addition of avocado significantly attenuated the rise in blood insulin levels by 37% and 22%, respectively (P = 0.04). Avocados are rich in antioxidants (e.g. polyphenolic compounds), which others have shown to be effective in improving insulin sensitivity in an overweight cohort [20]. Hence, including or adding avocado to a dietary pattern may assist in ameliorating the postprandial dysfunction in glucose homeostasis that may be present in overweight individuals. The AUC(0-3h) for blood insulin was lower in the AI test meal compared to both the C and the AA test meals, however this biological parameter did not significantly influence the five appetite sensation measurements between the AI and avocado-free C test meal (P = 0.07 to P = 0.64). It is worth noting that the five appetite sensation measurements for both the AI and AA test meals went in a favorable and similar direction, and borderline significant findings were found between the AI and C test meal in the context of increased satisfaction (P = 0.07) and a tendency existed towards reducing the desire to eat (P = 0.16). Insulin and the incretin hormones covary in response to elevated postprandial glucose levels [21], which makes it challenging to uphold the glucostatic theory proposed by Mayer [22]. Andersen et al. have observed that postprandial levels of blood glucose are inversely associated with self-reported appetite and food intake [23], however others have shown no association between satiety and blood glucose levels using an intravenous carbohydrate infusion [24]. Thus, it is plausible that the incretin hormones were influenced by the fat and fiber from the addition of avocado to the AA test meal, which yielded an increase in satisfaction and a reduction in the desire to eat. Although fat delays gastric emptying, some studies have shown that protein in the diet has the most potent action on satiety followed by carbohydrate, and fat the least [25,26]. However, it is important to note that studies designed to evaluate the satiety level of fat usually add fat to a meal in the form of oil or shortening, which increases the energy density of the meal without appreciably altering the volume of the meal. Thus, the low satiating effect of fat found in some studies may have been mediated exclusively by the increase in energy density. It is also worth noting that the intake at the ad libitum dinner and evening snack was similar between the AI and AA lunch test meals, and that the inclusion of avocado at a meal along with a concurrent reduction in other foods containing similar macronutrients favorably reduced the subsequent energy intake by 83 kcal (6.5%), and reduced the protein, carbohydrate and fat intake by 3.4 g, 6.0 g and 4.9 g, respectively. Avocados are a rich source of monounsaturated fatty acids, which are preferentially oxidized and increase thermogenesis as compared to polyunsaturated and saturated fatty acids. Thus, the inclusion of avocados to a dietary meal pattern may have additional implications in weight management in an overweight population. Although we found a significant reduction in insulin levels and favorable changes in two specific measures of appetite sensation for the AI and AA lunch test meals, respectively, we did not observe any behavioral change in dietary intake at the subsequent ad libitum dinner meal and evening snack between the 3 test meals. However, this latter null finding should not be over-interpreted as the data presented in this study are for 3 separate days (one week apart) and additional dietary energy compensation is plausible over several days and weeks [27]. This study had several strengths and limitations. Our controlled "laboratory" type setting had high internal validity due to the high degree of sensitivity and control over the dietary intervention and study outcome measures. An additional strength is that we analyzed the AUC appetite sensation data as opposed to a single time point because analysis of individual time points is not physiologically independent and is prone to type 1 errors. A study limitation is that we did not measure dietary intake in-between the 3 assigned study days. An additional study limitation is that we provided a wide variety of foods at our ad libitum dinner buffet meal, which is at variance with the typical eating pattern of most individuals and is likely to delay satiation and facilitate increased food intake [28]. Lastly, we may have placed the participants in an atypical environment by not providing food to them for 5 hours. BODY.CONCLUSIONS: This study showed that the addition of ~ 1⁄2 of a fresh Hass avocado to a lunch meal favorably increased satisfaction and reduced the desire to eat over a subsequent 3 hour and 5 hour period in an overweight and moderately obese adult population. When avocados were either added or included to a lunch meal, similarities in five measures of appetite sensation were found over a subsequent 3 hour period. Given that the peak satisfaction effect was found to be within 3 hours of the lunch test meal, subsequent studies should address the offering of snacks as this may be of importance to overweight and moderately obese adults that typically consume large snacks between meals. Therefore, the addition of ~ 1⁄2 of an avocado at a specific meal(s) may be a simple dietary intervention to consider for individuals that consume large snacks (e.g. excessive energy) between meals. Both the inclusion and addition of avocado to a lunch meal attenuated the rise in postprandial blood insulin levels 30 minutes after the start of the lunch meal as compared to the avocado-free control lunch meal. Additionally, the inclusion of avocado to a lunch meal yielded a significant reduction in blood insulin levels over a 3 hour postprandial period. The attenuation in the rise of insulin in the avocado inclusive intervention is worthy of future exploration in persons with insulin resistance and type 2 diabetes mellitus to determine if avocado intake can favorably influence measures of glucose homeostasis. Lastly, a longer trial would be beneficial to evaluate the effects of daily avocado intake on measures of appetite sensation and weight management in free-living normal weight, overweight and obese adults. BODY.COMPETING INTERESTS: The authors have no conflicts of interest or competing interests. BODY.AUTHORS’ CONTRIBUTIONS: EH and JS designed the study. EH, MW and JS coordinated the study. MW and EH were responsible for data collection, analysis and quality control. MW, EH, JS and KO were involved in the statistical analyses. All authors contributed to the interpretation of data. MW wrote the first draft of the manuscript and all authors critically reviewed and revised the manuscript. JS obtained the funding for the study. All authors read and approved the final manuscript.

TITLE: The effects of two different frequencies of whole-body vibration on knee extensors strength in healthy young volunteers: a randomized trial ABSTRACT: The aim of this study was to investigate the effects of two different frequencies of whole-body vibration (WBV) training on knee extensors muscle strength in healthy young volunteers. Twenty-two eligible healthy untrained young women aged 22-31 years were allocated randomly to the 30-Hz (n=11) and 50-Hz (n=11) groups. They participated in a supervised WBV training program that consisted of 24 sessions on a synchronous vertical vibration platform (peak-to-peak displacement: 2-4 mm; type of exercises: semi-squat, one-legged squat, and lunge positions on right leg; set numbers: 2-24) three times per week for 8 weeks. Isometric and dynamic strength of the knee extensors were measured prior to and at the end of the 8-week training. In the 30-Hz group, there was a significant increase in the maximal voluntary isometric contraction (p=0.039) and the concentric peak torque (p=0.018) of knee extensors and these changes were significant (p<0.05) compared with the 50-Hz group. In addition, the eccentric peak torque of knee extensors was increased significantly in both groups (p<0.05); however, there was no significant difference between the two groups (p=0.873). We concluded that 8 weeks WBV training in 30 Hz was more effective than 50 Hz to increase the isometric contraction and dynamic strength of knee extensors as measured using peak concentric torque and equally effective with 50 Hz in improving eccentric torque of knee extensors in healthy young untrained women. BODY.INTRODUCTION: Recently, whole-body vibration (WBV) training has been widely used in gyms, sports medicine, and rehabilitation clinics with potential beneficial effects for improving muscle strength and power, balance, bone health, or pain[1]. Indeed, two meta-anaylses evaluating the effects of WBV on muscular strength and power provided evidence that WBV was more effective in improving muscle strength and power in terms of knee extensor muscle strength and jumping performance when compared with controls without WBV[2] and similarly effective in enhancing chronic muscle power adaptations when compared with traditional plyometric exercises[3]. However, these meta-analyses[2,3] included/pooled studies with diverse populations (men and/or women, trained and/or untrained participants and/or athletes with differing ages) as well as with discrepant treatment protocols in relation to the type of vibration platform, either vertical or side-to-side alternating, exercise performed on the vibration platform (static and/or dynamic), the frequency and amplitude of WBV, the amount or duration of treatment, and the measurement methods. Along with this, while overall results were encouraging with regard to muscle strength and power, subgroup analyses yielded varying results in terms of participant profiles (young or middle-aged) and lower (≤30 Hz, <2-4 mm) or higher (>30 Hz-up to >4-6 mm) WBV frequencies and amplitudes[2]. The results of more recent studies (not included in the aforementioned meta-analyses[2,3]) on muscle performance in healthy adults also varied. Some studies indicated no significant effect of WBV on knee extensor muscle strength despite an increase in knee flexor muscle strength with vibration stimuli of 25 Hz and 6 mm on a side-to-side alternating vibration device[4] and with 40 Hz and 2-4 mm5 when added to resistance training with 20-40 Hz and 2.5-5 mm on a vertical vibrating platform[6]. Some others demonstrated significant improvements in jump performance with 30-45 Hz of frequency and 4 mm of peak-to-peak displacement on a vertical vibration platform[7] and with 18-32 Hz frequency and 1-3 mm amplitude[8]. Another study found an increase in knee extensor peak torque performance with a 40 Hz frequency and 0.76 peak-to-peak displacement on a vertically vibrating platform[9]. It is apparent that the results of studies on the effects of WBV on muscle strength and power are inconsistent thus far, not allowing inferences on the optimal WBV frequency for achieving significant muscle strength gain. Since differing vibration frequencies are known to be significant predictors of neuromuscular responses[1], it is important to determine which vibration frequency is better than the other for improving muscle strength. However, studies comparing different vibration frequencies for this purpose are not as many as those utilizing specific frequencies and focus on acute effects of WBV in the majority. In acute WBV effect studies (one bout of application) utilizing vibration frequencies and amplitudes ranging from 20 to 50 Hz and 2 to 5 mm (constant in most studies), while some studies favored 30 Hz frequency[10-12], some others favored frequencies within the range of 30-35 Hz[13], 40 Hz[14], or 50 Hz[15,16] for eliciting peak muscle activity, jump performance, or muscle strength and power when compared with other frequencies regardless of vibration amplitude in healthy young, middle-aged or older men and women. Adams et al.[17], comparing WBV stimuli of 30, 35, 40, and 50 Hz with amplitudes of 2-4 mm vs. 4-6 mm pointed to the effectiveness of higher vibration frequencies combined with higher amplitudes and lower frequencies combined with lower amplitudes for eliciting peak muscle performance in young males and females. Among the few long-term WBV training studies comparing different frequencies, while Petit et al.[18] reached the same conclusion with Adams et al.[17], the results of the study by Chen et al.[8] were in contradiction with those of Adams et al.[17] indicating higher frequencies with lower amplitudes (32 Hz, 1 mm) and lower frequencies with higher amplitudes (18 Hz, 3 mm) and with similar acceleration both led to improvement in jumping performance after 8 weeks of training. Thus, the impact of different frequencies of vibration on lower extremity muscle strength and power is still unclear and the debate seems to revolve around WBV frequencies between 30 Hz and 50 Hz. Based on all the aforementioned issues, there is a need for more research using different vibration frequencies over longer WBV training periods to be conducted on healthy people to identify which vibration frequency is better than the other to improve knee extensors muscle strength. The aim of the current study was to examine the effects of an 8-week WBV training program with two different frequencies of synchronous vertical vibration platform (30 and 50 Hz) with the same peak-to-peak displacement on isometric contraction and concentric/eccentric peak torques of knee extensor muscles in young healthy untrained women, and to compare the difference between 30 Hz and 50 Hz to provide more evidence for optimizing a WBV training protocol. BODY.MATERIALS AND METHODS.STUDY DESIGN: This study was a prospective randomized 8-week interventional trial, which was conducted between December 2011, and December 2012, at the Department of Physical Medicine and Rehabilitation in the Faculty Hospital. The study protocol was approved by the Faculty's Local Ethics Committee in conformity with the Declaration of Helsinki. All participants provided written informed consent before participating in the study after they were informed of the purpose of the study. BODY.MATERIALS AND METHODS.PARTICIPANTS: Twenty-two eligible healthy untrained young women aged between 22-31 years volunteered to participate in the study. The exclusion criteria were as follows: (i) prior experience of WBV training, (ii) participation in any systematic training programs that encompass strengthening and/or aerobic exercises at least 2 days per week, (iii) history of operation, severe trauma, and/or fracture, (iv) presence of musculoskeletal diseases such as acute herniated disc or spondylolisthesis, (v) presence of any condition that would complicate participation in exercise training programs (e.g. osteoarthritis in hip or knee joints), (vi) presence of malignancies, cardiovascular, pulmonary, gastrointestinal, urogenital, neuromuscular and/or any chronic diseases that might affect neuromuscular performance (e.g. diabetes mellitus, epilepsy), (vii) use of any medication that affects neuromuscular performance, (viii) presence of gall or kidney stones, prosthesis, and intraocular lens, (ix) smoking and alcohol consumption, and (x) pregnancy. Demographic characteristics of the participants were obtained at baseline assessment. BODY.MATERIALS AND METHODS.RANDOMIZATION: By the order of application to the department, 22 eligible participants were randomly allocated equally to the two groups using the computer-generated random numbers: 30-Hz Group (n=11) and 50-Hz Group (n=11). BODY.MATERIALS AND METHODS.DYNAMOMETRIC MUSCLE STRENGTH ASSESSMENT: In this study, strength of knee extensors was measured using Biodex Multi-joint System 3 Pro (Biodex Medical Systems, Inc., Shirley, New York, USA). To obtain more accurate results, all tests were performed at the same time of day (±1 h) by an experienced physiotherapist. During the test sessions, the temperature and humidity of the examination room was 21°C and 40%, respectively. All measurements were made unilaterally on the knee extensor muscles of the right lower extremity which was practically regarded as the dominant leg based on the right-handedness of all the participants shown to be in agreement with right-footedness in nearly 90% of women[19]. For all isokinetic tests, the participants were placed in a seated position with a trunk-thigh angle of 105° and strapped securely around the chest and waist to minimize movement of the trunk. During the testing procedure, each participant kept their arms crossed on their chest to minimize upper body influence on torque output. The rotation axis of the dynamometer was aligned with the knee flexion-extension axis on lateral condyle of the femur and the leg was strapped to the dynamometer lever arm 2 cm proximal to the lateral malleolus. For familiarization, all participants performed three habituation tests. The participants were also motivated with verbal encouragement throughout all tests to ensure that each test was performed with maximal effort. The participants were tested within 2 weeks of the beginning and the end of the study. To avoid any acute effect of training sessions on test results, post training measures were conducted at least 72 h after the last WBV training session. The testing procedure was performed under two conditions: isometric and dynamic contractions of the knee extensors. Isometric strength: The participants were examined for maximal voluntary isometric contraction of the knee extensors at 60° knee joint flexion (where 0° corresponded to full knee extension). Two trials (3-second duration) separated by a 60-second rest interval between attempts were completed and the highest value was recorded as maximal voluntary isometric contraction (Nm). Dynamic strength: Concentric and eccentric peak torques of knee extensors were evaluated at an angular velocity of 60°/s during isokinetic knee extension and flexion between 10° and 90° of knee angles. The participants performed a series of four consecutive isokinetic knee flexion and extension movements against the lever arm of the dynamometer. The greatest peak torque (Nm) for each of the concentric and eccentric contractions was used for analysis. BODY.MATERIALS AND METHODS.VIBRATION TRAINING: All participants were advised to continue normal daily living and maintain their current physical activity levels and asked not to participate in any systematic exercise training programs throughout the study. Training sessions: The WBV training program for both groups consisted of 20-60-minute sessions on three non-consecutive days per week (Monday, Wednesday, and Friday) for eight weeks. Each exercise session was strictly supervised by the investigators at the Department of Physical Medicine and Rehabilitation. Attendance of the participant was recorded by the investigators and was analyzed at the end of the study. All volunteers who participated in at least 80% of the WBV training sessions were defined as being adherent to the WBV training program and were included in the final analysis. In each WBV training session, the participants firstly performed a standardized 5-minute warm-up program consisting of lower extremity stretching exercises (primarily for quadriceps) and cycling on a stationary bike (50 watts) and then they were subjected to a WBV training program (see details below). Finally, they were trained to cool down with lower extremity stretching exercises (primarily for quadriceps) for about 10 minutes. The WBV training program consisted of unloaded static exercises. The WBV training volume was increased systematically over the 8 weeks by increasing the amplitude of vibration, the exposure time to vibration, the number of repetition for each exercise, and/or the number of different exercises. The duration of WBV training session, including warm up and cool down, was between 20-60 minutes, depending on the stage of the study. Details of the WBV training program are described in [Table 1]. Table 1 The volume and intensity of the WBV training program. Parameters 1-2 weeks 3-4 weeks 5-6 weeks 7-8 weeks LFG HFG LFG HFG LFG HFG LFG HFG Vibration frequency (Hz) 30 50 30 50 30 50 30 50 Peak-to-peak displacement (mm) 2 2 2 2 4 4 4 4 Peak acceleration (g) 3.6 10.0 3.6 10.0 7.6 20.1 7.6 20.1 Exposure to vibration without rest (s) 30 30 30 30 45 45 45 45 Repetition of each exercise (n) 2 2 4 4 6 6 8 8 Rest period between repetitions (s) 30 30 30 30 45 45 45 45 Total exposure to WBV in each session (min) 2 2 4 4 13.5 13.5 18 18 Different exercises which performed on the WBV platform  - Semi-squat + + + + + + + +  - One-legged squat + + + + + + + +  - Lunge - - - - + + + + WBV, whole-body vibration; LFG, low frequency group; HFG, high frequency group . Vibration conditions: A tri-planar (mostly vertical, Z axis) oscillating vibration platform (Power Plate® pro5TM; Power Plate North America, Inc., Northbrook, IL, USA) was used in all training sessions for generating mechanical vibration. The frequency of vibration platform was fixed at 30 Hz for the 30-Hz Group and 50 Hz for the 50-Hz Group throughout of the study. We used the predetermined setting of "low amplitude" (peak-to-peak displacement of the vibration platform: 2 mm) in the first four weeks, and "high amplitude" (peak-to-peak displacement of the vibration platform: 4 mm) during the second four weeks of the study similarly for both groups (Table 1). All participants were familiarized with the vibration platform and the correct positioning for use. During all training sessions, the participants stood on the vibration platform with sport socks (without shoes) and the foot position was standardized for all participants. Exercise positions: The participants in the two groups performed the following 3 unloaded static exercises that targeted knee extensor muscles: Semi-squat position: The participants stood on the vibration platform while the hands were placed on their waist and the trunk-thigh angle was 130°. Knee angles were 110° and 90° in the first and second halves of the study, respectively.One-legged squat position: The participants stood on the vibration platform using their right lower limb with their arms positioned such that their hands were holding the handlebars and the right knee angle was 90°. During vibrating periods, the left lower limb was not placed on the ground.Lunge position: The right foot was placed on the vibration platform with the right knee angled at 90°. The left foot was placed on the ground and the handlebars were permitted to be held to support their balance.Reporting of the study was made according to the recommendations of the International Society of Musculoskeletal and Neuronal Interactions[20]. BODY.MATERIALS AND METHODS.DATA ANALYSIS: The normality of distribution was determined by the Shapiro-Wilk test. Since all measures were normally distributed, the parametric tests were used for all statistical analyses. The homogeneity between the two groups was assessed using the independent-samples t test. To compare the posttest – pretest changes within each group, we used the paired-samples t test for two related samples. Improvement percent [(posttest group mean – pretest group mean) / (pretest group mean) x 100] was calculated for each group and compared by the independent-samples t test. To estimate effect size, the Cohen's d value was determined on the basis of the calculated t values. A p-value of <0.05 was considered as the significance level. All analyses were conducted using the SPSS software, version 17.0 for Windows (Statistical Package for the Social Sciences, Chicago, IL, USA). BODY.RESULTS.PARTICIPANT CHARACTERISTICS AND PROGRAM ADHERENCE: Demographic characteristics of the participants at beginning of the study are shown in [Table 2]. No significant difference existed between the two groups in age (p=0.451) and height (p=0.650) at the baseline. However, there was a significant difference between the two groups in terms of weight (p=0.036) and body mass index (p=0.031). Table 2 The homogeneity of demographic characteristics of the participants at baseline between the two groups. 30-HZ Group (n=10) 50-HZ Group (n=9) Mean ± SD (min-max) Mean ± SD (min-max) p * Age (years) 23.8 ± 2.7 (22-31) 24.7 ± 2.7 (22-31) 0.451 Height (cm) 165.1 ± 5.4 (153-172) 166.2 ± 5.1 (160-172) 0.650 Weight (kg) 64.2 ± 6.7 (49-74) 57.8 ± 5.1 (51-66) 0.036 BMI (kg/m 2 ) 23.5 ± 2.1 (20.7-27.5) 21.1 ± 2.3 (17.5-24.3) 0.031 SD, Standard Deviation; BMI, Body Mass Index. * Independent-samples t test. Of the 22 participants who participated in the training sessions, 19 individuals (10 in the 30-Hz and 9 in the 50-Hz groups) attended at least in 20 of the 24 training sessions (83.3%). The three participants (1 in the 30-Hz and 2 in the 50-Hz groups) who had an attendance rate below 80% were excluded from the final analysis (see Table 3). No significant difference existed between the two groups in the attendance rates (p=0.286). Table 3 The attendance rate of the participants in vibration training programs and the comparison between the two groups. Dropped-out Included in the study Number of Completed Sessions, Maximum n. 24; n (%) < 20 (83.3) 20 (83.3) 21 (87.5) 22 (91.6) 23 (95.8) 24 (100) p * Number of participants  30-Hz Group (Total n. 11) 1 2 1 3 1 3 0.286  50-Hz Group (Total n. 11) 2 3 2 2 1 1 * Independent-samples t test (n.19). BODY.RESULTS.OUTCOME MEASURES: There was no significant difference between the two groups at the baseline assessment in terms of all outcome measures (p>0.05). The maximal voluntary isometric contraction significantly increased in the 30-Hz Group (+20.3%; p=0.014); whereas, the slight increase in the 50-Hz Group (+2.6%; p=0.445) was not found statistically significant (see Table 4) with a significant between-group difference (p=0.039). The Cohen's d effect size was 1.08 for maximal voluntary isometric contraction. Table 4 Changes in knee extensors strength from the baseline to the 8-week by the training groups. Outcome measures Mean ± SD Change Within-group Between-groups Group Baseline 8-week (%) p † p †† Cohen’s d ††† Isometric strength, MVIC (nm) 30-Hz 113.7 ± 26.1 136.6 ± 17.0 +20.3 0.014 0.039 1.08 50-Hz 119.3 ± 14.7 122.5 ± 22.8 +2.68 0.445 Dynamic strength, concentric PT (nm) 30-Hz 107.2 ± 15.1 121.8 ± 17.7 +13.6 <0.001 0.018 1.27 50-Hz 108.4 ± 16.7 110.7 ± 9.8 +2.1 0.605 Dynamic strength, eccentric PT (nm) 30-Hz 104.2 ± 17.4 119.9 ± 22.6 +16.0 0.001 0.873 0.07 50-Hz 93.3 ± 16.2 108.3 ± 13.6 +16.0 0.003 SD, standard deviation; MVIC, maximal voluntary isometric contraction; PT, peak torques. † Paired-samples t test . †† Independent-samples t test . ††† Based on the t values . Similarly, over 8 weeks, a statistically significant increase has occurred in the concentric peak torque of knee extensors in the 30-Hz (+13.6%; p<0.001) group, while not in the 50-Hz (+2.1%; p=0.605) group. When the groups were compared, there was a significant (p=0.018) difference between the two groups in terms of improvements in the concentric peak torque. The Cohen's d effect size was 1.27 for concentric peak torque. As seen in [Table 3], there was a significant increase in the eccentric peak torque of knee extensors in both the 30-Hz (+16.0%; p=0.001) and the 50-Hz (+16.0; p=0.003) groups after the 8-week WBV training program without any significant difference between the two groups (p=0.873). The effect size (Cohen's d) was 0.07 for eccentric peak torque. BODY.DISCUSSION: In the current research, we examined the impact of two different vibration frequencies on knee extensors muscle strength in untrained healthy young volunteers. Taken together, we concluded that the supervised 8-week WBV training program in 30 Hz of vibration frequency was more effective than 50 Hz in increasing knee extensors strength in terms of maximal isometric contraction and peak concentric torque and equally effective with 50 Hz in terms of peak eccentric torque. Our finding demonstrating better effectiveness of a WBV frequency of 30 Hz on increasing muscle strength is not surprising and is highly supported by other WBV studies. Acute WBV studies (using a similar WBV device) suggested greater effectiveness of WBV frequency of 30 Hz than those of 20 or 40 Hz (with an amplitude of 4 mm)[10] and 35, 40 or 50 Hz (with amplitudes of 2 and 5 mm)[11] and with a low amplitude (2-4 mm) (specifically when measured at 10 minutes post exposure)[17] in improving jumping performance, a muscle power variable shown to be significantly associated with maximal strength[21] in young untrained or moderately trained men and women. Cardinale and Lim[22] found the frequency of 30 Hz as generating the highest muscular electromyographic activity in a half-squat position on a sinusoidal vertical vibration platform allowing up to 10 mm peak-to-peak displacement in female volleyball players when compared with WBV frequencies of 40 Hz, 50 Hz, and sham WBV. The same situation applied to physically active middle-aged men and women in whom 30 Hz of vibration frequency on a side alternating platform produced the highest muscle electromyographic activity with a significantly greater increase than that produced by 6 Hz or 12 Hz; however, without any significant difference between other tested frequencies of 18 and 24 Hz[12]. Furthermore, Da Silva-Grigoletto et al.[23] pointed to the effectiveness of 30 Hz WBV frequency with an amplitude of 4 mm in improving jumping performance in young men. Contrarily, Ronnestad[15] and Marin et al.[16], again using vertically vibrating platforms, proposed a vibration frequency of 50 Hz with amplitudes of 3 mm and 2.51 mm, respectively, as more beneficial than 20 and 35 Hz in improving 1 RM squatting performance[15] and as more effective than 30 Hz with an amplitude of 1.15 mm in increasing muscle activity as measured using electromyography[16]. It is important to note that acute effect studies favoring 50 Hz included heavily loaded dynamic exercises such as squatting either on the vibration platform[15] or before exposure to vibration[16]; whereas, none of the other acute effect studies favoring 30 Hz involved dynamic heavily loaded exercises other than cycle-ergometer, range of motion or stretching exercises for warm-up before vibration exposure[10,11,17,22,23] as was the case also in our study. A very recent study that demonstrated improvement in quadriceps strength after dynamic squats and a decrease in strength after static squats with WBV (synchronous vibrating platform, set at 30 Hz and 4 mm amplitude) in untrained young males and females[24] may well provide an explanation on why 30 Hz of vibration frequency in studies by Ronnestad[15] and Marin et al.[16] was inferior to 50 Hz of frequency which was in an advantageous position for increasing squat performance in trained and/or untrained males and females due to the greater thigh muscle stimulating capability of higher frequencies than that of lower frequencies during squatting[15]. Proceeding to long-term studies, Petit et al.[18], comparing the effects of 6-week WBV training programs with different frequency and peak-to-peak displacement (30 Hz/2 mm versus 50 Hz/4 mm) also demonstrated that high-frequency/high peak-to-peak displacement WBV training was more effective than low frequency/low peak-to-peak displacement WBV or sham training to increase the eccentric knee extensor muscle strength, thus indicating superiority of 50 Hz of vibration frequency to 30 Hz, however, with only statistically significant difference from baseline when compared to 30 Hz in eccentric knee eccentric peak torque (increasing by 16.3%) and not in either isometric strength or concentric peak torque with regard to isokinetic testing. It was interesting to note that we also found 16.0% increase from baseline in the peak knee extensor eccentric torque, however, in both groups. The discrepancy between our results and those of the study by Petit et al.[18] regarding other isokinetic testing parameters might be explained by several factors: differing number of repetition of exercise on the vibration platform (starting from 2 and increasing to 8 in our study vs 10 repetitions in their study) and duration of vibration exposure (between 2 and 18 minutes in each session vs 10 min in each session), demonstrated to have been important in strength gain[23]. Additionally, the difference between the amplitude of vibration, knee angle (with a significant influence on strength gains[12,25,26]) and the profile of the participants [physically active but nonresistance-trained young males (physical education students) in the study by Petit et al.[18] and young untrained females in our study] may play a role in this difference. On the other hand, another long-term study by Martinez-Pardo et al.[27] did find that WBV training with stimuli of 50 Hz and 4 mm significantly increased concentric torque of knee extensors after 6 weeks of WBV training with a working time of 60s and 60s rests and with incremental training of 8 to 13 sets of exercises with a synchronous vibrating platform in recreationally active young men and women, contrary to the findings of Petit et al.[18] who observed no significant changes in concentric torque. Yet in another long-term study, Chen et al.[8] demonstrated that an 8-week WBV training with high vibration frequency and low amplitude (32 Hz and 1 mm) and with low vibration frequency and high amplitude (18 Hz and 3 mm) with the same acceleration both led to significant increases in jumping performance in moderately trained young adults, attributable to different neuromuscular adaptations[8], including 'tonic vibration reflex[28], proprioception, and body control[29] and balance[8]. Therefore, the study by Chen et al.[8] partly supported our finding of strength gain with a vibration frequency of 30 Hz in a long-term study. On the contrary, de Ruiter et al.[30] reported that WBV training of the same frequency but with a different amplitude (30 Hz, 8 mm) had no effect either on maximal voluntary isometric contraction of knee extensors or on countermovement jump height in healthy students after 11 weeks. The design of our study does not allow valid comments on the mechanism of why 30 Hz did a better job than did 50 Hz because we did not assess WBV induced changes on muscular activity or other contractile properties of the muscles. Proposed mechanisms include reflex contraction of muscles, the so-called 'tonic vibration reflex' associated with Ia afferents of the muscle spindle, increased responsiveness of Golgi tendon related Ib afferents influencing muscle length, neuromuscular facilitation including increased motor unit recruitment, synchronization, proprioception, coordination, better co-contraction of synergist muscles, increased ability of the muscles to generate force due to hypergravity, enhancement of muscle perfusion, and muscle hypertrophy[1,28,31]. We may speculate that mechanisms other than 'tonic vibration reflex' might be at play in our study due to the notion that the effect of this mechanism is mostly seen in combined dynamic strength training and WBV protocols and at higher frequencies particularly in athlete populations[31]. Whatever the mechanisms may be, the capability of 30 Hz of vibration to increase electromyographic activity of knee extensors has been shown in immediate effect studies[12,28] and its effect on knee extensor strength is also supported by other long-term studies demonstrating effectiveness of frequencies as little as 24-28 Hz with 2-4 mm of amplitude in increasing concentric torque of knee extensors when added to resistance training better than resistance training alone even in competitive skiers[32]. Indeed, effect sizes as calculated using Cohen's d in our study could be considered large for isometric contractions and concentric peak torque with d values of 1.08 and 1.27, respectively, (Table 4) indicating a nonoverlap of estimably more than 50% in the 30 and 50 Hz groups based on the descriptions relevant to Cohen's d as d=0.8 indicating a nonoverlap of 47.4. However, Cohen's d effect size of 0.07 for eccentric peak torque showed that 30 Hz and 50 Hz groups did not differ as also reflected by independent samples t-test (p=0.873)[33]. The reason why isometric contraction and concentric peak torque showed significantly greater increase with 30 Hz vibration frequency and eccentric peak torque did not remains to be elucidated in larger sample sized studies including control groups. It is apparent that the effect of WBV training program on muscle strength is a matter of vast debate in many aspects, particularly with regard to frequency. In our study, we demonstrated a significant increase in the maximal voluntary isometric contraction of knee extensors, concentric and eccentric peak torque of knee extensors over 8 weeks with a vibration frequency of 30 Hz. While there was a trend to improve in isometric contraction and concentric torque of knee extensors with 50 Hz, the strength gain did not reach a statistical significance; however, 50 Hz of vibration frequency with amplitude incrementally increasing from 2 mm to 6 mm did significantly increase the eccentric peak torque of knee extensors. We believe that our study contributes to evidence in WBV research by providing an efficient protocol that had a progressive nature because the intensity of all variables including vibration conditions, set numbers, and types of static exercises on the platform increased systematically throughout the study. Although it has recently been shown in an acute effect study that dynamic squats on a vibration platform with a frequency of 35-40 Hz was associated with increased quadriceps force production contrary to static squats leading to decreased strength in young men and women indicated not having performed resistance exercises within the past year[24], we observed the opposite, that is, muscle strength increasing effects of WBV training with static squats with either 30 Hz or 50 Hz. However, we cannot tell if static squats had an additive effect to WBV training or vice versa due to the methodology of the study. Siu et al.[34] also found immediate knee extensor concentric peak torque changes with 26 and 40 Hz vibration frequency with peak-to-peak displacements 8 mm and 3.38 mm which were significantly greater than that in the controls exercising static half-squats on a side alternating vibration platform. Advantageous aspects of the combination of static squats with WBV training could be the rapid increase in muscle temperature (for recommending as a warm-up exercises) and less oxygen demand (oxygen uptake) when compared to the combination of dynamic squats with WBV (difference being not statistically significant) as found in a study in young active men and women having exercised on a side alternating platform with a frequency of 26 Hz[35]. Another study found the lowest heart rate response and peak oxygen uptake in sedentary young males after WBV training with static squats than in those after WBV training with dynamic or loaded squats with a vibration frequency of 32 Hz on an oscillatory vibration platform[36], which may have implications for better tolerability, however, with less beneficial effects on aerobic capacity. The demonstration of the effectiveness of 30 Hz of vibration frequency with incrementally increasing amplitudes from low (2 mm) to high (4 mm) on increasing all isokinetic test variables is important based on the findings that this frequency was shown to cause significantly lower rate of perceived exertion as well as lower heart rate compared to 50 Hz[37], allowing its prescription to untrained individuals who may have difficulty in continuing a WBV training program or other exercise programs because of their high demand. Moreover, high frequencies have been found associated with greater muscle fatigue[38] as well as impaired balance as reflected by center of pressure velocity and length, having been found higher in vibration frequencies of 40 and 44 Hz than those of 31 and 35 Hz with approximately 1 mm amplitude on a vertical synchronous vibration device[39]. BODY.DISCUSSION.STRENGTH AND LIMITATION OF THE STUDY: Our study had a number of strengths. A major strength of this study was the relatively long period of WBV training (8 weeks). While quite a number of studies have investigated the acute effects of different vibration frequencies as discussed previously[10-17,34,37], the number of studies that compared the effects of different vibration frequencies for a long period of time is limited[8,18,27]. Secondly, in the current study we included healthy untrained volunteers who have not been exercising more than two times a week. This is important considering that most previous investigations on the effectiveness of WBV training have included moderately active/recreationally trained females and/or males[4,8,11,14,16,27], even physical education students[18], or men and/or women participating regularly in sports[10], except a few including both trained and untrained individuals[15] or only untrained participants[17]. In terms of maintaining good attendance, it is difficult to conduct a training program on healthy and untrained individuals multiple times per week for several weeks who, possibly, are not that interested to improve their musculoskeletal condition. Another strength of the study was the progressive property of WBV training program within 8 weeks in terms of vibration conditions (see Table 1). Furthermore, we prescribed exercises in different knee angles such as one-legged squat and lunge positions to increase the impacts of vibration on the knee extensor muscles, specifically (see Table 1). Additionally, in the current study, all WBV training sessions were supervised by investigators. Finally, the relatively high rate of attendance at WBV training sessions also gave strength to the study. Despite these strengths, our study had a number of limitations that should be considered when interpreting the results. First and most notable, the sample size of the study was too small. Second, the lack of control group was another major limitation of the study. It should be considered that application of really sham vibration on participants is very hard and partly inapplicable. In conclusion, the findings of this study indicated that 30 Hz of vibration frequency was more effective than 50 Hz in increasing of isometric and concentric peak torque of knee extensor muscles after 8 weeks of WBV training and equally effective with 50 Hz in increasing eccentric torque of knee extensors in young healthy volunteers. Additionally, the contents of the present training protocol can be used to produce optimal recommendations for effective WBV training programs in clinical practice and training centers for improving the strength of knee extensors particularly in untrained individuals who might prefer easier and less strenuous training. However, there is still a need to undertake more randomized controlled studies with different vibration conditions and a larger number of participants to confirm these results.

TITLE: Effect of Inpatient Multicomponent Occupational Rehabilitation Versus Less Comprehensive Outpatient Rehabilitation on Sickness Absence in Persons with Musculoskeletal- or Mental Health Disorders: A Randomized Clinical Trial ABSTRACT: Purpose To assess effects of an inpatient multicomponent occupational rehabilitation program compared to less comprehensive outpatient rehabilitation on sickness absence in persons with musculoskeletal- or mental health disorders. Methods Randomized clinical trial with parallel groups. Participants were individuals 18–60 years old on sick-leave for 2–12 months with a sick-leave diagnosis within the musculoskeletal, psychological or general and unspecified chapters of ICPC-2, identified in a national register. The inpatient program (4 + 4 days) consisted of Acceptance and Commitment Therapy (ACT), physical training and work-related problem-solving including creating a return to work plan and a workplace visit if considered relevant. The outpatient program consisted primarily of ACT (6 sessions during 6 weeks). Both programs were group based. Primary outcome was cumulated number of sickness absence days at 6 and 12 months follow-up. Secondary outcome was time until sustainable return to work. Results 168 individuals were randomized to the inpatient program (n = 92) or the outpatient program (n = 76). We found no statistically significant difference between the programs in median number of sickness absence days at 6 and 12 months follow-up. In the outpatient program 57% of the participants achieved sustainable return to work (median time 7 months), in the inpatient program 49% (log rank, p = 0.167). The hazard ratio for sustainable return to work was 0.74 (95% CI 0.48–1.32, p = 0.165), in favor of the outpatient program. Conclusions This study provided no support that the more comprehensive 4 + 4 days inpatient multicomponent occupational rehabilitation program reduced sickness absence compared to the outpatient rehabilitation program. BODY.INTRODUCTION: Too many people leave the workforce prematurely due to health problems or disability, and too few workers with health problems are able to stay in work [1], particularly due to musculoskeletal and mental health disorders [2]. In addition to individual suffering this causes considerable costs for society. In Norway 5% of the gross domestic product is spent on disability and sickness benefits [1]. Several treatment and rehabilitation programs to facilitate work participation have been investigated, notably for persons with low back pain, but also for common mental disorders [3–6]. However, previous studies have been performed in outpatient settings, whereas there is a long tradition for inpatient multicomponent occupational rehabilitation in Norway. These programs usually consist of cognitive behavioral therapy, physical exercise and patient education [7], but little workplace involvement [8]—which is considered important in improving return to work rates [9–11]. There is some support for including these components in such programs. Cognitive behavioral therapy is recommended for patients with chronic low back pain [12] and common mental health disorders [13], and is often included in return to work interventions [14]. Physical exercise provides substantial health benefits [15, 16] and is also inversely associated with disability pension [17] and sickness absence [18]. Patient education is considered beneficial in treatments of chronic low back pain [12] and common mental health disorders [19], and often included in return to work programs [14, 20]. Still, no randomized studies have assessed the effect of inpatient multicomponent occupational rehabilitation on work participation. The diagnosis-specific emphasis of previous studies [3–5, 9, 21], is somewhat in contrast to the increasing documentation of overlap between musculoskeletal complaints and mental health problems [22, 23], and the fact that return to work rehabilitation programs for low back pain also have been suggested to be useful for persons on sick leave with mental health disorders [24]. In line with this, occupational rehabilitation centers in Norway include different diagnostic groups in the same program [7]. However, we are not aware of studies evaluating return to work rehabilitation programs for both somatic and mental health disorders with a rigorous study design. Although inpatient occupational rehabilitation programs in Norway typically last about 4 weeks where patients live at the centers, there are several reasons for investigating different approaches: (1) in a pilot-investigation, several participants reported that 4 weeks was too long to stay away from home; (2) a continuous stay at a rehabilitation center does not allow for workplace involvement, and (3) a 4-week rehabilitation period is based on traditions rather than scientific evidence, and less costly alternatives should be investigated. Hence, we designed a randomized study investigating effects on sick leave of an inpatient multicomponent occupational rehabilitation program lasting 4 + 4 days, separated by 2 weeks where a workplace visit could be performed. The comparative program was a less comprehensive outpatient program, consisting mainly of a recent form of cognitive behavior therapy [25]. We hypothesized that the inpatient multicomponent occupational program would reduce sickness absence more than the less comprehensive outpatient program, as it in addition to cognitive behavioral therapy, included physical training, patient education, a return to work plan and a workplace visit when relevant. BODY.METHODS.STUDY DESIGN AND PARTICIPANTS: We conducted a randomized clinical trial with parallel groups, comparing an inpatient multicomponent occupational program with a single-component outpatient program (hereafter also referred to as the inpatient- and outpatient program, respectively) for individuals on sick-leave due to musculoskeletal-, unspecific-, or common mental health disorders. Details about the study design have been published in a protocol article [8]. The study was approved by the Regional Committee for Medical and Health Research Ethics in Central Norway (No.: 2012/1241), and the trial is registered in https://clinicaltrials.gov/ (No.: NCT01926574). The results are presented according to the CONSORT statement [26]. Eligible participants were 18 to 60 years of age sick listed 2 to 12 months with a diagnosis within the musculoskeletal (L), psychological (P) or general and unspecified (A) chapters of the ICPC-2 (International Classification of Primary Care, Second edition). The current sick leave status had to be at least 50% off work. Exclusion criteria, assessed by a comprehensive questionnaire and an outpatient screening performed by a physician, physiotherapist and psychologist, were: (1) alcohol or drug abuse; (2) serious somatic (e.g. cancer, unstable heart disease) or psychological disorders (e.g. high suicidal risk, psychosis, ongoing manic episode); (3) specific disorders requiring specialized treatment; (4) pregnancy; (5) currently participating in another treatment or rehabilitation program; (6) insufficient oral or written Norwegian language skills to participate in group sessions and fill out questionnaires; (7) scheduled for surgery within the next 6 months; and (8) serious problems with functioning in a group setting. BODY.METHODS.INTERVENTIONS: The inpatient program consisted of several components; group-based cognitive behavioral therapy, individual and group-based physical training, mindfulness, psychoeducation on stress and individual meetings with the coordinators for work-related problem-solving and creating a return to work plan. The cognitive behavioral approach was Acceptance and commitment therapy (ACT), which is a new form of cognitive behavioral therapy that emphasizes accepting both negative and positive experiences while using the individuals` values to guide them towards their goals [25]. Studies have suggested an effect of ACT on the main causes of sickness absence, namely chronic pain [27], anxiety [28] and depression [28, 29]. Through mindfulness techniques, values and committed action the aim of ACT is to increase psychological flexibility [30] and to increase return to work by increasing coping and motivation, as indicated by a randomized pilot study [31]. ACT was chosen as the cognitive behavioral therapy-approach in this study because of its transdiagnostic approach [32]. The intervention lasted four full workdays in week 1 and week 4 (8 days in total; 6–7 h each day) during which the participants resided at the rehabilitation center, separated by 2 weeks at home (week 2 and 3). The 2 weeks at home included at least two contacts with the team coordinator (in person or by telephone) and a meeting with the employer if regarded relevant and the participant gave permission. The coordinators who mentored the participants were supervised by a certified ACT-instructor before and during (monthly) the intervention. The program took place at Hysnes rehabilitation center, established as part of St. Olavs Hospital, in central Norway. The outpatient program consisted primarily of one component; group-based ACT. The sessions were held at the Department of Physical Medicine and Rehabilitation at St. Olavs Hospital once a week for 6 weeks, each session lasting 2.5 h. The sessions were led by either one of two physicians (specialists in Physical medicine and rehabilitation) or a psychologist; all supervised by the same ACT instructor as the coordinators in the inpatient program. The participants were given assignments to practise at home between sessions, including a daily 15 min audio-guided mindfulness practice. In addition the participants were offered two individual sessions with a social worker experienced in occupational rehabilitation and trained in ACT to clarify personal values and work-related issues. The program also included a motivational group discussion with a physiotherapist on the benefits of physical training. An individual session with both the social worker and group leader present ended the program. In this session a summary letter was written to the participant's general practitioner. BODY.METHODS.STUDY CONTEXT: All legal residents in Norway are included in the Norwegian public insurance system. Medically certified sick leave is compensated with 100% coverage for the first 12 months. The first 16 days are covered by the employer, the rest by the Norwegian Welfare and Labour Administration. After 12 months of sick leave it is possible to apply for the more long-term medical benefits, work assessment allowance and disability pension, which both covers approximately 66% of the income. Individuals on work assessment allowance are supposed to work according to their workability. BODY.METHODS.OUTCOME MEASURES: Participants were followed for 12 months after inclusion. During this period, sickness absence was registered in monthly intervals, both as number of days per month and as a dichotomous measure of whether or not the participant was registered on sick leave that month. Outcomes were measured using data from the National Social Security System Registry, where all individuals receiving any form of benefits in Norway are registered by their social security number. The data consisted of registrations of medical benefits from four different sources; sick-leave payments, sick leave certificates, work assessment allowance and disability pension. Monthly intervals (rather than exact dates) were used in order to include all relevant sick leave benefits in the same measure, as exact dates were not available for payments and the long-term benefits. Work assessment allowance was adjusted for delay in payments up to 2 months. The primary outcome measure was cumulated number of sickness absence days, calculated at 6 and 12 months after inclusion. By combining information from the different medical benefits we calculated days on medical benefits (according to a 5-day work week) for every month during follow-up. Time on graded sick leave was transformed to whole workdays. Days receiving sick-leave payment and work assessment allowance were adjusted for employment fraction, including a graded disability pension at inclusion. Any increase in disability pension during follow-up was counted as sick leave. The secondary outcome measure was time until full sustainable return to work defined as 1 month without relapse, i.e. one monthly interval not receiving any medical benefits (except any graded disability the participant had when entering the study). Questionnaires measuring baseline characteristics like education, pain, anxiety and depression symptoms were answered by the participants before the screening. Anxiety and depression were assessed using The Hospital Anxiety and Depression Scale (HADS) [33]. It consists of 14 items, where seven items measure anxiety and seven depression symptoms. It is scored on a 4-point Likert scale according to intensity of symptoms in the last week. The maximum score is 21 on each subscale. To assess pain we used one question from the Brief Pain Inventory [34]. The participants were asked to grade the average pain during the last week on a 0 (no pain) to 10 (worst imaginable pain) numeric rating scale. BODY.METHODS.RANDOMIZATION: Invited participants completed a short questionnaire assessing initial eligibility. Those eligible were invited for an outpatient screening assessment. If the screening was passed (Fig. 1), subjects were randomized to either the inpatient or the outpatient program. A flexibly weighted randomization procedure was provided by the Unit of Applied Clinical Research (third-party) at the Norwegian University of Science and Technology, to ensure that the rehabilitation center had enough participants to run monthly groups in periods of low recruitment. This affected group-sizes differentially, and therefore the researchers were not blinded. Sickness absence data was registered and provided by employees at the Norwegian Welfare and Labor Service whom were unaware of group allocation. It was not possible to blind neither the participants nor the caregivers for treatment. Fig. 1Participant flow through the study BODY.METHODS.SAMPLE SIZE: Sample size calculations were based on three approaches [8]: Comparison of RTW with Kaplan Meier survival analysis with log rank test with a hazard ratio of 0.6 (alpha 0.05, beta 0.20) would require 63 in each group.Comparison of number of days with sick leave at 6 months of follow-up (p = 0.05; 90% power): An average of 60 days (SD 40) and 90 days (SD 60) of sick leave in the intervention and comparative group, respectively would require 61 persons for each group.Comparing ratios of participants at work after 1 year of follow-up with the same statistical assumptions as point 2; and a difference of 60 versus 40% RTW, would require 63 people in each group. With an estimated 20% loss to follow-up we aimed to include 80 persons in each arm. The sample size calculations were based on results from previous studies in this field [5, 9, 10]. BODY.METHODS.STATISTICAL ANALYSIS: Number of days of sick leave at 6 and 12 months after inclusion for the two programs were calculated and compared using the Mann–Whitney U (Wilcoxon rank sum) test. For time until sustainable return to work Kaplan Meier curves were estimated and compared with the log rank test. We estimated hazard ratios for return to work using Cox proportional hazard model with the Efron method for ties [35]. Time was calculated as number of months and participants were censored at "full sustainable return to work" or end of follow-up. We performed analyses without adjustment and with adjustment for gender, age, level of education, main diagnosis for sick leave and length of sick leave at inclusion. The proportionality hazard assumption was checked using the Schoenfeld Residual Test [36]. All analyses were performed after the "intention to treat" principle. Additional "per protocol" analyses were done by excluding participants that withdrew after randomization (before or during the programs) and/or attended less than 60% of the sessions of the outpatient program. In addition to the main analyses, we performed several post hoc sensitivity analyses in order to account for characteristics of the sickness absence patterns and data structure which we observed in the course of the study. First, we observed that several participants alternated between being on and off benefits. We therefore performed a repeated events analysis allowing individuals to alternate between being on and off benefits every month of follow-up using general estimating equations (GEE). Secondly, we observed single months without payment in between longer periods of payments. As the Norwegian holiday lasts 5 weeks, we performed an additional sensitivity analysis on time until sustainable return to work where we defined return to work as 2 months without benefits. We considered p-values (two-tailed) <0.05 to be statistically significant. Precision was assessed using 95% confidence intervals. All analyses were done using STATA 13.1 (StataCorp. 2013. Stata Statistical Software: Release 13. College Station, TX: StataCorp LP). BODY.RESULTS: The flow of participants through the study is illustrated in Fig. 1. Between October 2012 and November 2014, 12 007 potential participants from the regional area were identified in the National Social Security System Registry and 3 318 were randomized to receive an invitation to the short program. Of these 275 accepted the invitation. After screening 168 remained and were randomized to the inpatient program (n = 92) or the outpatient program (n = 76). The groups consisted of maximum 9 participants. For the inpatient program, 14 people withdrew before they began the program and four quit during the program. For the outpatient program, five people withdrew before the program started and eight during the program. Those who started the outpatient program attended on average 7.9 of the 10 meetings and 59 (83%) attended at least 60% of the sessions. For the inpatient program there is no data available regarding the number of sessions participants attended, but as it was an inpatient program the participants were assumed compliant if they did not withdraw. All participants were included in the analyses. A workplace visit was performed for 13% (n = 10) of the participants who started the inpatient program. BODY.RESULTS.PARTICIPANTS` CHARACTERISTICS: Most of the participants (65%) worked full time prior to their sick-leave, while 18% worked part time, 4% had a graded disability pension and 13% had no job. The median number of days on sick-leave the last 12 months before inclusion in the study (i.e. second randomization) was 226 calendar days (interquartile range (IQR) 189–271). A musculoskeletal diagnosis was most common (52%), followed by psychological (38%) and general and unspecific (10%) diagnoses. The mean age of participants was 45 years and the majority was women (79%). The baseline characteristics of the participants in the two programs were fairly similar (Table 1). Table 1Baseline characteristics of participantsInpatient program (n = 92)Outpatient program (n = 76)Age mean (SD)45.0 (8.7)45.1 (9.6)Women n (%)71 (77%)62 (82%)Higher education n (%)a 45 (49%)31 (41%)Work status n (%) No work15 (16%)7 (9%) Full time57 (62%)52 (68%) Part time15 (16%)16 (21%) Graded disability pensionb 5 (5%)1 (1%)Sick-leave status n (%)c Full sick-leave41 (45%)35 (46%) Partial sick-leave45 (49%)36 (47%) Work assessment allowance6 (7%)5 (7%)HADS mean (SD) Anxiety (0–21)7.8 (4.4)7.4 (4.3) Depression (0–21)6.7 (4.3)6.0 (4.1)Pain level mean (SD) Average pain (0–10)4.7 (2.3)4.6 (2.0)Main diagnoses for sick-leave (ICPC-2) n (%)c A—general and unspecified9 (10%)7 (9%) L—musculoskeletal48 (52%)40 (53%) P—psychological35 (38%)29 (38%)Length of sick leave at inclusionc,d Median days (IQR)224 (189–262)229 (187–275) aHigher (tertiary) education: college or university bIndividuals working part time that at inclusion also received a graded disability pension cBased on data from the National Social Security System Registry dNumber of days on sick leave during the last 12 months prior to inclusion. Measured as calendar days, not adjusted for partial sick- leave BODY.RESULTS.SICKNESS ABSENCE DAYS: The median number of sickness absence days (work days) at 6 months after inclusion was 58 (IQR 37–92) for the inpatient program and 51 (IQR 32–85) for the outpatient program. The difference was not statistically significant (Mann–Whitney U test, p = 0.284). For the 12 months follow-up, the median number of sickness absence days was 114 (IQR 46–172) for the inpatient program and 96 (IQR 35–175) for the outpatient program (Fig. 2). The difference was not statistically significant (Mann–Whitney U test, p = 0.403). Fig. 2Cumulative number of days (median) on medical benefits for the inpatient- and the outpatient program during 12 months of follow-up. Adjusted for employment fraction and transformed to whole workdays according to a 5-day work week BODY.RESULTS.SUSTAINABLE RETURN TO WORK: In total 88 participants achieved sustainable return to work (i.e. 1 month without benefits) during 12 months follow-up, 45 participants (49%) in the inpatient program and 43 participants (57%) in the outpatient program. Median time until sustainable return to work was 7 months for the outpatient program (IQR 4-not reached). The inpatient program did not reach 50% return to work in the follow-up period (IQR 5-not reached). Figure 3 shows the Kaplan Meier plot. The difference between the programs was not statistically significant (log rank test: p = 0.167). Cox regression analysis without adjustment gave a hazard ratio of 0.74 (95% CI 0.48–1.32, p = 0.165) for sustainable return to work, in favor of the outpatient program. Adjustment for age, gender, education, main diagnosis for sick leave and length of sick leave at inclusion gave similar results (hazard ratio 0.72, 95% CI 0.46–1.11, p = 0.135). Fig. 3Survival curves from the Kaplan Meier analysis showing time to sustainable return to work (i.e. 1 month not receiving medical benefits) for the inpatient- and the outpatient program BODY.RESULTS.OTHER SICKNESS ABSENCE MEASURES: Of the participants achieving sustainable return to work, 15 participants (33%) in the inpatient program and 20 (47%) participants in the outpatient program returned to medical benefits during the 12 months follow-up. At 12 months, 40 participants (43%) in the inpatient program and 30 (39%) in the outpatient program was not on medical benefits (excluding graded disability benefits). About half the participants received work assessment allowance in both groups (50 and 49% respectively) and 5% of the participants in the inpatient program and 12% in the outpatient program were on sick leave. One participant in the inpatient program received full disability pension. Repeated events analyses for return to work showed no difference between the programs at any of the time points (months of follow-up) (Fig. 4). The average odds ratio over time was 0.78 (95% CI 0.49–1.24, p = 0.299) for return to work (i.e. 1 month without benefits) in favor of the outpatient program. Adjusting for aforementioned variables did not change the conclusion. Fig. 4Estimated probabilities of not receiving benefits at each month during 12 months of follow-up. Results from a repeated events analysis using logistic general estimating equations (GEE) When the analyses were performed using 2 months without medical benefits as event, the sustainable return to work rate dropped slightly to 45 and 53% for the inpatient and outpatient program respectively. Unadjusted and adjusted cox regression gave hazard ratios similar to the analyses performed with1 month without benefits as the event. Per protocol analyses comparing number of sickness absence days in the inpatient and outpatient programs showed similar results as the main analyses at 6 months: 60 (IQR 39–96) versus 53 (IQR 32–82; p = 0.187) days and at 12 months: 118 (IQR 48–181) versus 98 (IQR 39–157; p = 0.313) days. The per protocol cox regression analyses also showed similar results as the main analyses: unadjusted HR 0.71 (95% CI 0.44–1.16, p = 0.174), and adjusted HR 0.70 (95% CI 0.43–1.15, p = 0.161). BODY.DISCUSSION: Among persons on sick leave with a musculoskeletal, psychological or unspecific diagnosis, this randomized trial showed no significant difference in number of sickness absence days and time to sustainable return to work following an inpatient multicomponent occupational rehabilitation program compared to a less comprehensive outpatient program. Even though there were no statistical differences between the programs, there were some indications that participants in the outpatient program had less sickness absence days and shorter time to sustainable return to work. However, this group also had a higher fraction of recurring sickness episodes. Hence, we performed a post hoc analysis to assess the probability of receiving/not receiving monthly medical benefits throughout the 1-year follow-up period. Assessing sickness absence in this way made the between-group differences smaller, strengthening the finding of no difference between the programs. Return to work rates in this study were lower than in some previous return to work studies [3, 5, 6]. However, those studies only included participants with musculoskeletal complaints while this study also included common mental health disorders and unspecific complaints. The participants in this study also had longer current sickness episodes than some of the previous studies [3, 6], which might indicate more complex problems. They were also invited directly through the National Social Security System and not referred by a physician. Nevertheless, the low return to work rate for the inpatient program could indicate that the program did not match their needs. Studies have suggested that involving the workplace in return to work programs is effective for reducing sick leave for individuals on sick leave with low back pain [5, 9] and common mental health disorders [4]. The inpatient program in this study involved one workplace visit, but only when considered relevant by the participant and the rehabilitation team, and was only performed for 13% of the participants. The reasons for not performing the workplace visit were poorly registered. Focus group interviews with individuals participating in a similar but more long-lasting program at the same rehabilitation center found that few had made concrete plans for return to work at the end of the program [37]. That so few workplace visits were performed could possibly in part explain why there was no additional effect of the inpatient program compared to the outpatient program. In a Norwegian context this was a relatively short inpatient occupational rehabilitation program (4 + 4 days), as traditional inpatient programs typically last about 4 weeks [7]. In that regard, lack of difference between the two programs could be due to the short length of the inpatient program. As the participants included in the study had median sick leave duration of more than 200 days in the year before inclusion, they might have needed a longer rehabilitation program to facilitate the return to work process. Similarly, we cannot exclude the possibility that the outpatient program potentially might have been more effective, had it been more comprehensive. However, we are not aware of studies showing added effect of more intensive programs [38, 39]. The main strength of this randomized study was the use of registry data on medical benefits, ensuring that there were no biased assessments of end-points and no missing data. Furthermore, all participants were invited from the National Social Security System, meaning there was no referral bias. However, there was a self-selection bias as to which individuals accepted the invitation to participate in the study. Accepting the invitation meant they had to be prepared to be away from family and friends during the program if allocated to the inpatient program. From more than 3000 invitations sent, only 275 individuals accepted the invitation, which limits the generalizability of the results. Even though an inclusion criterion was sick leave for at least 8 weeks, the mean length of sick leave at inclusion was more than 220 days for both programs. It could be that individuals with greater obstacles for return to work to a larger extent accepted the invitation. This assumption is strengthened by the fact that around 50% of the participants received work assessment allowance at 12 months follow-up. As this medical benefit provided after 1 year of sickness absence only reimburses 66% of the salary compared to 100% for sick leave pay, there is a considerable financial incentive for returning to work within 1 year. As there is no randomized usual care control group we do not know if the programs reduced sick leave and increased return to work compared to usual care. Another limitation was that the researchers were not blinded. However, sickness absence was registered and provided by employees at the Norwegian Welfare and Labor Service whom were unaware of group allocation. BODY.CONCLUSION: Among persons on sick leave with a musculoskeletal, psychological or unspecific diagnosis, this study provides no support that the 4 + 4 days inpatient multicomponent occupational rehabilitation program reduces sickness absence compared to a less comprehensive outpatient program. As the inpatient program was more resource-demanding we do not recommend it to be implemented in regular clinical practice. Considering that this program was relatively short in an inpatient setting, future studies should investigate effects of more extensive inpatient occupational rehabilitation programs.

TITLE: Do research findings on schema-based instruction translate to the classroom? ABSTRACT.INTRODUCTION: Schema-based instruction has been shown to improve diagnostic performance and reduce cognitive load. However, to date, this has only been studied in controlled research settings. More distractions in classrooms may limit generalizability to real-world settings. We evaluated whether schema-based instruction would maintain its effects on cognitive load optimization and performance in a classroom. ABSTRACT.METHODS: Focused on the approach of interpreting cardiac auscultation findings, 101 first-year medical students at Western University were randomized to receive a traditional (n = 48) or a schema-based lecture (n = 53). Students completed four written questions to test diagnostic performance and a cognitive load assessment at the end of the lecture. Diagnostic performance and cognitive load were compared with independent t-tests. ABSTRACT.RESULTS: Schema-based instruction was associated with increased diagnostic performance on written questions (64 ± 22 % vs 44 ± 25 % p < 0.001) and reduced intrinsic cognitive load (mean difference = 15 %, standard error 3 %, p < 0.001). There was no significant difference in reported extraneous (p = 0.36) or germane (p = 0.42) cognitive load. ABSTRACT.CONCLUSIONS: Our results demonstrate that schema-based instruction can be used to reduce intrinsic load and improve diagnostic performance in a real-world classroom setting. The results would be strengthened by replication across other locations and topics. BODY.INTRODUCTION: Applying principles from cognitive psychology to instructional design in medical education has demonstrated improvements in performance [1]. Within research studies, applications of cognitive psychology have been associated with increased diagnostic performance [2–6]. However, studies exploring the translation of beneficial interventions to the classroom are limited, despite criticism over the generalizability of research results to practical teaching settings. In medicine, schemas are described in relationship to diagnoses. In this context, a schema is a diagnostic algorithm that organizes a differential diagnosis by the presenting complaint and provides clinically relevant discriminating features to distinguish similar diagnoses (Fig. 1; [5]). In contrast, traditional instructional formats organize information by diagnosis, often in lists of unlinked facts.Fig. 1Schema used in schema-based lecture Patients typically present with an undifferentiated clinical problem rather than a known diagnosis. Information provided in a schema is more conducive to assessing undifferentiated complaints as it is organized according to the presenting complaint; however, the information in a traditional format must be reorganized. Providing information in traditional formats emphasizes comprehensiveness, whereas organization into a schema focuses the learner on the features to distinguish diagnoses. Presenting a differential diagnosis in a schema encourages comparing and contrasting between diagnoses, mirroring the decision-making process required when assessing patients. The improved diagnostic accuracy associated with schema use in research settings is well described [2–6]. Improvements of up to 35 % in diagnostic accuracy on practical tasks and 20 % on written questions have been observed with schemas [2]. Cognitive load theory (CLT) can be used to explain the improved diagnostic performance seen with schemas. CLT is based on the premise that working memory is limited [7]. Cognitive load can be optimized by addressing each of its subcomponents: intrinsic, extraneous and germane cognitive load. Intrinsic load describes the inherent difficulty of the task. Schemas optimize intrinsic load by reducing the total number of facts, grouping similar information and organizing information in a clinically relevant format. Extraneous cognitive load refers to any mental effort that does not contribute to learning. It is ideally minimized. Schemas may decrease extraneous load by organizing the information around the presenting complaint, avoiding the reorganization of information required if provided by individual diagnoses. Germane cognitive load describes the processing required to transfer information from working memory to storage in long-term memory. Schemas optimize germane cognitive load by chunking information and associating similar diagnoses. Schema-based instruction has been associated with lower overall cognitive load as compared with traditional methods of instruction [6]. Prior studies have evaluated interventions under research protocols with volunteer subjects. The controlled designs may limit generalizability of the results to a classroom setting. There may be more distractions in a classroom, such as internet resources, classmates or mobile technology [8]. As such, classrooms may have higher extraneous load than research conditions. There is limited evidence supporting the application of CLT to the classroom. Issa et al. [9] studied the translation of multimedia design principles into clerkship lectures, finding improved written performance when students were instructed with lectures that optimized multimedia design and cognitive load. In another small study, lectures modified to optimize cognitive load were associated with higher knowledge scores and lower cognitive load as compared with traditional lectures [10]. In an observational study, participants who used a schema for metabolic disturbances when answering written questions had higher diagnostic accuracy scores. [3]. It is unclear how the increased extraneous cognitive load of a classroom environment impacts beneficial effects of schema-based instruction. The potential interactions between intrinsic, extraneous and germane load have been debated [11, 12]. The increased extraneous load in the classroom could consume working memory and negate any potential reductions in intrinsic load [12]. Alternatively, the benefits of schemas may be accentuated with increased extraneous load because the optimized intrinsic load requires less working memory. Distractions could have a greater impact on traditional methods of teaching that require more working memory. The purpose of this study was to evaluate whether schema-based instruction would maintain its effects on cognitive load and diagnostic performance in a classroom setting. We hypothesized that the schema-based lecture would decrease intrinsic load and increase diagnostic performance. BODY.METHODS.PARTICIPANTS AND PROCEDURE: The study was embedded within a lecture on structural heart disease in the Cardiology curriculum for first-year medical students at Western University. The participants had competed 5 months of medical school on topics unrelated to Cardiology prior to the study. We utilized a single-blind randomized controlled trial design. Students were randomized using a random number generator. Both lectures were designed around the same objectives: describe and classify normal heart sounds, abnormal heart sounds and cardiac murmurs. Ten structural lesions (aortic stenosis, atrial septal defect, hypertrophic cardiomyopathy, tricuspid regurgitation, ventricular septal defect, mitral regurgitation, mitral valve prolapse, aortic regurgitation, mitral stenosis and pericardial rub) were described. The lectures contained the same number of slides (81 slides) with the same average number of words per slide (21 words). Both lectures were given in a standard 1 hour lecture time frame in lecture halls used in the undergraduate medical curriculum at Western University. The lectures were given simultaneously by two different lecturers. Both lectures began with a review of the physiology of normal heart sounds, followed by a discussion of abnormal heart sounds. To ensure that no group was significantly disadvantaged prior to the course examination, both lectures were repeated immediately following data collection. BODY.METHODS.CONTROL LECTURE: The details of each lesion were listed in succession. The lecturer outlined the character, radiation, extra sounds, and palpable findings for each murmur. A junior faculty member who had provided this lecture in previous years delivered the lecture. BODY.METHODS.EXPERIMENTAL LECTURE: The schema-based lecture presented the same introduction. Murmurs were presented according to timing and location using an adaptation of a published schema (Fig. 1; [2]). For example, when systolic murmurs at the base of the heart were described, a differential diagnosis of aortic stenosis, atrial septal defect and hypertrophic cardiomyopathy was presented. The lecturer then explained the pathophysiology and associated findings that would allow differentiation between the differential provided. The lecture was given by a junior lecturer who had used this schema in previous research studies [2]. BODY.METHODS.INSTRUMENTS: Immediately following the lecture, participants completed a cognitive load assessment about the lecture and a four-item test assessing diagnostic performance. The cognitive load assessment was based on a previously validated tool [13] to assess the subcomponents of cognitive load. Each subcomponent consisted of 3–4 individual questions on a 10-point scale. Subcomponent scores were added to arrive at a score for intrinsic (maximum 30 points), extraneous (maximum 30 points) and germane (maximum 40 points) cognitive load. The four written multiple-choice questions were generated by three experts in cardiac auscultation. The questions provided details of the cardiac physical exam. Participants provided the diagnosis without the aid of any instructional materials. Three weeks following the study date, participants rated the lecturers on a 7-point scale (1 = below expectations, 4 = average, 7 = outstanding). BODY.METHODS.STATISTICS: Independent t-tests were performed to compare the cognitive load assessments and written questions. Lecturer ratings were compared with a paired t-test. Ethics approval was granted by the Western University Research Ethics Board. BODY.RESULTS: Of 133 students, 101 participated (76 %), with 48 randomized to the traditional lecture and 53 randomized to the schema-based lecture. Schema-based instruction was associated with increased performance on the diagnostic performance test (64 % ± 22 vs 44 % ± 25, p < 0.001). Participants rated intrinsic cognitive load lower with the schema-based lecture (20 vs 24 points, mean difference = 15 %, standard error 3 %, p < 0.001). There was no significant difference in reported extraneous (11 vs. 12 points, mean difference = 3 %, standard error = 3 %, p = 0.36) or germane (22 vs 21 points, mean difference = 3 %, standard error = 4.5 %, p = 0.42) load. Fifty-nine (59 %) participants evaluated the lecturers. Participants rated both lecturers similarly (traditional lecture: 4.98 vs schema-based lecture: 5.25, p = 0.114). BODY.DISCUSSION: Mirroring findings from research-oriented settings, we identified an increase in diagnostic performance and a reduction in intrinsic cognitive load with schema-based instruction in a classroom setting. We observed an increase in diagnostic performance of 20 % in the schema group. This effect size is comparable to a small study of lectures optimized using cognitive load theory (15 %) [10]. This effect size is also in keeping with a study that identified an increase in written diagnostic accuracy of 20 % with the use of a similar schema for murmur identification conducted in research settings [2]. The similarity of effect sizes suggests that the potential increased extraneous load of the classroom setting did not undermine the optimized intrinsic load of the schemas. The reduction in intrinsic load may reflect the different organizational approaches for the traditional and schema-based lectures. The schema-based lecture focused on discriminating facts and contextualized pathophysiological explanations for abnormal cardiac findings into discussion of differential diagnosis, whereas the traditional lecture focused on describing the conditions. While the reduction in intrinsic load could also be attributed to lecturer attributes, the similarities between the two lecturer ratings argue that the organizational approach likely had more impact on the intrinsic load measurements. The similar extraneous load measurements support the argument that performance differences were due to the organizational approach to the material rather than differences in the lecturer's style or different amounts of classroom distractions. There are several limitations to this study. Firstly, it was conducted at a single centre and results would be strengthened by replication at other sites. Secondly, two different lecturers provided the lectures and differences in style could have influenced the results. We attempted to control the effect of lecturer style by controlling slide content. Thirdly, the outcome measure for diagnostic performance was limited to four questions and not standardized; however no standardized questions on cardiac auscultation exist in the literature. Lastly, delayed performance was not measured. Providing the complementary lecture following the study lecture limited our ability to conduct delayed testing. BODY.CONCLUSION: In conclusion, schema-based instruction maintained its beneficial effects in a classroom environment. Our findings support the translation of applied educational theory to the classroom.

TITLE: Adenosine A ABSTRACT: BackgroundWe evaluated the efficacy and safety of istradefylline, a selective adenosine A2A receptor antagonist administered as adjunctive treatment to levodopa for 12 weeks in a double-blind manner in Parkinson's disease patients with motor complications in Japan. MethodsA total of 373 subjects were randomized to receive placebo (n = 126), istradefylline 20 mg/day (n = 123), or istradefylline 40 mg/day (n = 124). The primary efficacy variable was the change in daily OFF time. Other secondary variables were also evaluated. ResultsThe change in daily OFF time was significantly reduced in the istradefylline 20 mg/day (−0.99 hours, P = .003) and istradefylline 40 mg/day (−0.96 hours, P = .003) groups compared with the placebo group (−0.23 hours). The most common adverse event was dyskinesia (placebo, 4.0%; istradefylline 20 mg/day, 13.0%; istradefylline 40 mg/day, 12.1%). ConclusionsIstradefylline reduced daily OFF time and was well tolerated in Japanese PD patients with motor complications on levodopa treatment. BODY: Long-term levodopa treatment causes motor complications such as wearing-off and dyskinesias in Parkinson's disease (PD).1 Various therapeutic approaches have been developed to overcome these difficulties while maintaining adequate therapeutic levodopa levels. However, the complications and dopaminergic side effects of long-term levodopa treatment are not yet fully resolved.2 Istradefylline, a selective adenosine A2A receptor antagonist, is considered nondopaminergic because of the lack of effects on dopamine receptors and dopamine-metabolizing enzymes. Istradefylline is a new antiparkinsonian drug that can be added as a new treatment option to current PD therapy.3,4 In experimental parkinsonian animals, istradefylline, when used in combination with levodopa, exhibits an additive effect on motor control without worsening levodopa-induced dyskinesia.6–9 All10–14 but 115 of the previous studies showed a significant decrease in OFF time. A phase 2b Japanese study showed reduction in daily OFF time16; it is the purpose of this study to confirm our previous results. BODY.PATIENTS AND METHODS: The diagnosis of PD depended on the UK Parkinson's Disease Society Brain Bank criteria.17 Key inclusion criteria included at least 3 doses of levodopa/decarboxylase inhibitor per day (daily dosage of 300 mg), a stable regimen of all antiparkinsonian drugs for at least 4 weeks prior to randomization, at least 2 hours of OFF time per day, and stages 2 to 4 on the modified Hoehn & Yahr stage (OFF state). Key exclusion criteria included a history of neurosurgery for PD, transcranial magnetic stimulation for PD within 6 months before randomization, dementia or a score of 23 or less on the Mini–Mental State Examination (MMSE), pregnant or lactating women, women planning to have children, and prior istradefylline exposure. Subjects were at least 20 years old when written informed consent was obtained. This study was a multicenter, placebo-controlled, randomized, double-blind, parallel-group, and confirmatory study conducted between July 2009 and February 2011 at 44 investigative Japanese sites with institutional review board approval based on the principles described in the Declaration of Helsinki. After giving informed consent, subjects were randomly assigned to receive istradefylline 20 or 40 mg/day or placebo orally once daily for 12 weeks. Subjects completed diaries for 7 consecutive days before visits in weeks 2, 4, 8, and 12. At each visit, physicians assessed subjects on the Unified Parkinson's Disease Rating Scale (UPDRS) Parts I–IV and treatment-emergent adverse events (TEAEs). Electrocardiograms (ECGs) were done every 4 weeks. At the end point, the modified Hoehn & Yahr stage and Clinical Global Impression–Improvement of illness (CGI-I) were assessed. Antiparkinsonian drugs were not changed during the 4 weeks prior to randomization. Reductions in the dosages of antiparkinsonian drugs were permitted only for TEAEs. The primary efficacy variable was the difference between the total hours of awake time per day spent in the OFF state (daily OFF time) from the baseline diaries and those collected at the last visit. The secondary efficacy variables included change in percentage of awake time per day spent in the OFF state, change in total hours of awake time per day spent in the ON state, change in percentage of awake time per day spent in the ON state, and changes in UPDRS Parts I–IV, CGI-I, and the modified Hoehn & Yahr stage. Safety assessments were the same as in the previous study.16 All efficacy analyses were conducted on the full analysis set (FAS), defined as subjects who received at least 1 dose of the study drug and submitted at least 4 valid diaries for evaluation for any assessment times after starting the study drug. The statistical methods used were essentially the same as in the previous study.16 This study (6002-009) was registered as ClinicalTrials.gov number NCT00955526. BODY.RESULTS: A total of 373 subjects were randomized as shown in Supporting Fig. 1. A total of 109 placebo, 111 istradefylline 20 mg/day, and 115 istradefylline 40 mg/day subjects completed 12 weeks of treatment. The numbers of patients who were prematurely withdrawn from the study are also shown in Supporting Figure 1. The FAS included 366 subjects (placebo, 123; istradefylline 20 mg/day, 120; and istradefylline 40 mg/day, 123); 7 subjects were excluded from the FAS because of missing 4 valid diaries. The Safety Set included all 373 randomized subjects. Fewer men were included in the istradefylline 20 mg/day group; more subjects used concomitant selegiline in the istradefylline 40 mg/day group; and fewer subjects used concomitant entacapone in the placebo group. Other demographics and characteristics were comparable (Table 1). Table 1 Demographic and baseline characteristics (full analysis set) Characteristic Placebo (n = 123) Istradefylline 20 mg/day (n = 120) Istradefylline 40 mg/day (n = 123) Age (y), mean (SD) 65.8 (8.6) 66.1 (8.6) 65.7 (9.0) Male, n (%) 58 (47.2%) 40 (33.3%) 64 (52.0%) BMI, mean (SD), kg/m 2 22.17 (3.59) 22.34 (3.40) 22.37 (3.65) Time since diagnosis (y), mean (SD) 7.990 (4.453) 7.301 (4.206) 7.730 (4.547) Time since onset of motor complications (y), mean (SD) 3.432 (3.470) 3.183 (2.759) 3.258 (3.009) Daily OFF time  Mean (SD), h 6.31 (2.47) 6.55 (2.72) 5.97 (2.45)  Mean (SD), % 38.91 (14.80) 40.59 (16.19) 36.92 (15.10) Daily ON time  Without dyskinesia (h), mean (SD) 8.53 (2.84) 7.93(3.38) 8.50(3.54)  With dyskinesia (h), mean (SD) 1.35(2.50) 1.57(2.75) 1.83(3.30)  With nontroublesome dyskinesia (h), mean (SD) 0.94 (1.95) 1.00 (1.71) 1.13(2.03)  With troublesome dyskinesia (h), mean (SD) 0.41 (1.11) 0.58 (1.63) 0.69 (1.75)  Without troublesome dyskinesia (h), mean (SD) 9.47 (2.54) 8.93 (2.86) 9.64 (2.82) UPDRS Part I subscale score, mean (SD) 1.1 (1.5) 1.2 (1.4) 1.0 (1.4) UPDRS Part II subscale score (ON state), mean (SD) 5.9 (5.2) 5.3 (5.2) 5.3 (5.0) UPDRS Part II subscale score (OFF state), mean (SD) 14.7 (7.7) 14.9 (7.5) 15.4 (8.1) UPDRS Part III subscale score (ON state), mean (SD) 21.6 (11.6) 21.3 (10.8) 20.7 (11.0) UPDRS Part IV subscale score, mean (SD) 4.7 (2.0) 5.1 (2.2) 5.0 (2.4) Daily dosage of prior levodopa (mg), mean (SD) 425.4 (146.4) 430.8 (156.5) 420.5 (131.8) Concomitant antiparkinsonian medications, n (%)  Dopamine agonists 112 (91.1%) 103 (85.8%) 103(83.7%)  Anticholinergic agents 20 (16.3%) 12 (10.0%) 19(15.4%)  Selegiline 57 (46.3%) 52 (43.3%) 75 (61.0%)  Entacapone 52 (42.3%) 63 (52.5%) 68(55.3%)  Amantadine 49 (39.8%) 41 (34.2%) 44 (35.8%)  Zonisamide 17 (13.8%) 13 (10.8%) 20 (16.3%) The changes from baseline at the end point for daily OFF time for placebo, istradefylline 20 mg/day, and istradefylline 40 mg/day were −0.23, −0.99 (P = .003), and −0.96 (P = .003) hours, respectively. The differences from the placebo were significant, but there were no difference between the 2 groups. The changes for secondary efficacy variable are shown in Table 2; daily ON time without troublesome dyskinesia for placebo, istradefylline 20 mg/day, and istradefylline 40 mg/day were 0.26, 1.09 (P = .003), and 1.08 (P = .004) hours, respectively. Neither istradefylline 20 mg/day nor istradefylline 40 mg/day increased daily ON time with troublesome dyskinesia. The changes from baseline at end point for UPDRS Part II score (OFF state) for placebo, istradefylline 20 mg/day, and istradefylline 40 mg/day were −0.6, −1.4 (P = .034), and −1.7 (P = .009), respectively. The changes from baseline at end point for UPDRS Part III score (ON state) for placebo, istradefylline 20 mg/day, and istradefylline 40 mg/day were −2.8, −3.7 (P = .086), and 4.9 (P = .001), respectively, showing that istradefylline 40 mg/day significantly reduced UPDRS Part III score (Table 2). The percentages of subjects who were "Much improved" plus "Very much improved" at end point for placebo, istradefylline 20 mg/day, and istradefylline 40 mg/day were 10.7%, 20.8% (P = .005), and 28.7% (P < .001), respectively (Supporting Fig. 2). No differences were observed among the groups for other secondary efficacy variables. Clinical variables such as age, sex, and others did not affect the effectiveness of istradefylline in reducing daily OFF time. Daily OFF/ON time and UPDRS subscale score—actual data and change from baseline values (full analysis set) Placebo (n =123) Istradefylline 20 mg/day (n =120) Istradefylline 40 mg/day (n =123) Actual Change Actual Change Actual Change Daily OFF time End point, LS mean (h) 6.05 −0.23 5.29 −0.99 5.31 −0.96  LS mean vs. placebo ( P ) — — — −0.76 (.003 a ) — −0.74 (.003 a ) End point, LS mean, % 37.24 −1.55 32.24 −6.55 32.62 −6.17  LS mean vs. placebo ( P ) — — — −4.99 (.002 a ) — −4.61 (.003 a ) Daily ON time Without dyskinesia End point, LS mean (h) 8.60 0.28 9.22 0.9 9.18 0.85  LS mean vs. placebo ( P ) — — — 0.61 (—) — 0.57 (.033 NS) With dyskinesia End point, LS mean (h) 1.51 −0.08 1.81 0.22 1.68 0.09  LS mean vs. placebo ( P ) — — — 0.30 (—) — 0.17 (.139 NS) With nontroublesome dyskinesia End point, LS mean (h) 0.98 −0.04 1.27 0.25 1.19 0.16  LS mean vs. placebo ( P ) — — — 0.29 (—) — 0.21 (.108 NS) With troublesome dyskinesia 0.50 −0.06 0.55 −0.01 0.54 −0.02 End point, LS mean (h) — — — 0.05 (—) — 0.04 (.421 NS)  LS mean vs. placebo ( P ) Without troublesome dyskinesia End point, LS mean (h) 9.61 0.26 10.44 1.09 10.42 1.08  LS mean vs. placebo ( P ) — — — 0.83 (.003 a ) — 0.81 (.004 a ) UPDRS Part I End point, LS mean 0.9 −0.2 1.0 −0.1 1.0 −0.1  LS mean vs. placebo ( P ) — — — 0.1 (—) — 0.1 (.906 NS) UPDRS Part II (ON state) End point, LS mean 5.2 −0.3 5.2 −0.3 5.0 −0.5  LS mean vs. placebo ( P ) — — — 0.0 (—) — −0.2 (.290 NS) UPDRS Part II (OFF state) End point, LS mean 14.4 −0.6 13.6 −1.4 13.4 −1.7  LS mean vs. placebo ( P ) — — — −0.8 (.034 NS) — −0.1 (.009 a ) UPDRS Part III (ON state) End point, LS mean 18.4 −2.8 17.5 −3.7 16.3 −4.9  LS mean vs. placebo ( P ) −0.9 (.086 NS) — −2.0 (.001 a ) UPDRS Part IV End point, LS mean 4.7 −0.2 4.8 −0.2 4.5 −0.4  LS mean vs. placebo ( P ) — — — 0.1 (—) — −0.2 (.213 NS) a P  < .025 ( P value by Williams test). Least squares (LS) mean and P values are based on the main effects ANCOVA with terms for baseline, investigator and treatment. NS, not significant. TEAEs occurred in 51.6%, 65.0%, and 59.7% of subjects in the placebo, istradefylline 20 mg/day, and istradefylline 40 mg/day groups, respectively. Dyskinesia was the most frequently reported TEAE in both istradefylline groups. The other TEAEs are shown in Supporting Table 1. One subject treated with placebo died on day 19. The situation was unknown at the time of death. Serious adverse events were observed in 2 subjects receiving placebo (2 events; toxicity to various agents and breast cancer in situ), in 6 subjects receiving istradefylline 20 mg/day (8 events; pneumonia bacterial, gait disturbance, radius fracture, neuralgia, sciatica, parkinsonism, delirium, and bile duct cancer), and in 6 subjects receiving istradefylline 40 mg/day (6 events; gastric ulcer, bronchitis, myocardial infarction, pneumonia aspiration, hallucination, and rectal cancer). Of these events, gait disturbance, parkinsonism, gastric ulcer, myocardial infarction, and hallucination were considered drug-related TEAEs. All events resolved or were alleviated. No clinically meaningful changes from baseline were observed in laboratory results, body weight, vital signs, ECG, or MMSE score. BODY.DISCUSSION: Adenosine A2A receptors in striatum are selectively localized on GABAergic output neurons of the striatopallidal pathway.3 Increase in GABA release from the globus pallidus after 6-hydroxydopamine injection into the medial forebrain bundle was reversed by systemic injection with istradefylline in rats.18 This study (6002-009) demonstrated the efficacy and safety of istradefylline in Japanese PD subjects. Istradefylline 20 and 40 mg/day significantly reduced daily OFF time with increases in daily ON time without troublesome dyskinesia compared with placebo. But no dose response was seen between 20 and 40 mg/day. This was probably because adenosine receptor occupancy in PET was reported to be more than 90% with a dose of adenosine 5 mg in healthy controls.19 These decreases in OFF time were independent of subject demographics. Istradefylline 40 mg/day significantly improved UPDRS Part II (OFF state) and Part III (ON state) scores compared with placebo. The most frequently reported TEAE was dyskinesia, which occurred with a higher incidence in subject treated with istradefylline than with placebo. All occurrences were mild or moderate in severity and were not dose dependent. Findings in this phase 3 study (6002-009) were consistent with the phase 2b study (6002-0608). These findings demonstrate that istradefylline administered as adjunctive therapy to levodopa reduced daily OFF time and further improved motor functions, thus showing a nondopaminergic drug that can be added to any existing PD therapy. To date, 4 confirmatory studies have been conducted in advanced PD in the United States using a design similar to this study. Three studies reported reduction in daily OFF time,12,13 but in 1 study, istradefylline did not provide significant improvement,15 possibly because of substantial placebo effect. Dyskinesia was the most frequent adverse event in all studies, with a lower incidence in Japan. The efficacy and safety data in this study indicate that oral istradefylline 20 or 40 mg once daily is effective in relieving wearing-off phenomena and further improving motor functions in Japanese PD patients. Therefore, istradefylline can be added on to other existing antiparkinsonian therapies.

TITLE: Comparative clinical efficacy of three toothpastes in the control of supragingival calculus formation ABSTRACT.OBJECTIVES:: The purpose of this double-blind, parallel clinical study was to assess clinical efficacy in supragingival calculus formation reduction using Abhaibhubejhr Herbal Toothpaste compared to Colgate Total and Colgate Cavity Protection toothpastes. ABSTRACT.MATERIALS AND METHODS:: A total of 150 subjects participated in the pretest phase. All subjects were given oral soft/hard tissue evaluation, calculus examination using Volpe-Manhold calculus, and whole mouth oral prophylaxis. They received noncalculus control fluoride toothpaste and a soft-bristled toothbrush to brush for 1 min two times daily for 8 weeks. After which, subjects were given a test phase oral soft/hard tissue evaluation and calculus examination and were randomized into one of the three toothpaste groups. All subjects in the test phase received a whole mouth oral prophylaxis and were given their assigned toothpaste and a soft-bristled toothbrush to brush for 1 min two times a day for 12 weeks. Thereafter, subjects were assessed for their oral soft/hard tissue and calculus formation. ABSTRACT.RESULTS:: Mean Volpe-Manhold calculus index scores for the Cavity Protection, Abhaibhubejhr, and Total toothpaste groups were 0.78, 0.62, and 0.48, respectively, at the 12-week test phase evaluation. Abhaibhubejhr and Total toothpaste groups show 20.51% and 38.46% significantly less calculus formation than the Cavity Protection toothpaste group (P < 0.05). Total toothpaste group also show 22.58% significantly less calculus formation than the Abhaibhubejhr toothpaste group (P < 0.05). ABSTRACT.CONCLUSION:: The use of Colgate Total toothpaste over a 12-week period was clinically more effective than either Abhaibhubejhr or Colgate Cavity Protection toothpastes in controlling supragingival calculus formation. BODY.INTRODUCTION: Dental calculus or tartar is calcified deposit occurring on the teeth or other solid structures in the oral cavity such as restorations, prosthetic appliances, and dental implants. Calculus is calcified dental plaque which can be classified as supragingival or subgingival calculus. Dental plaque is the diverse community of microorganisms found on tooth surface as a biofilm. The microorganisms bind tightly to one another and the tooth surface by means of an extracellular matrix of polymers of host and microbial origin.[12] Dental plaque biofilms are responsible for many common oral diseases including dental caries, gingivitis, periodontitis, and peri-implantitis.[3] Calculus is invariably covered with plaque on its surface. Calculus promotes the retention of dental plaque and may increase the rate of plaque formation. Its porosity can serve as a reservoir for periodontal pathogens and can retain noxious bacterial components such as endotoxin.[45] Recently, there has been a growing interest in natural products. People's interest has been shifted from synthetic toothpaste to herbal toothpaste. Herbal toothpaste does not contain any artificial colors or flavors that many of the regular toothpaste have. Artificial colors and flavors of the toothpaste are also giving birth to many diseases and health-related issues. The demand for herbal toothpaste suppliers has risen due to the great advantages which are associated with this toothpaste type. There are a number of brands of herbal toothpaste on the market. Abhaibhubejhr is a widely known herbal-based toothpaste containing Guava leaves, Betel vine leaves, and Mangosteem peel for which medicinal properties are claimed. However, many of the health claims made for them have not been clinically proven. The objective of this clinical study was to evaluate the anticalculus efficacy of Abhaibhubejhr Herbal Toothpaste compared to Colgate Total toothpaste (positive control) and Colgate Cavity Protection toothpaste (negative control). BODY.MATERIALS AND METHODS.SCREENING AND SELECTION OF SUBJECTS: This study was approved by the Faculty of Dentistry, Mahidol University Institutional Review Board (COA. No.MU-DT/PY-IRB 2012/016.1204). Adult subjects from the Faculty of Dentistry, Mahidol University, Thailand, signed an informed consent form and were screened by the examining dentist to identify those subjects who meet the inclusion characteristics and demonstrate a tendency to form calculus. The first 150 subjects who meet the inclusion/exclusion were entered into the pretest phase of this double-blind, randomized, parallel clinical study. Inclusion characteristics included good general health, male and female subjects, aged 18–70, inclusive, scoreable lower six anterior teeth free of large restorations or dental prosthetic crowns, a Volpe-Manhold calculus index score of at least 10, and available for the 20-week duration of the study. Exclusion characteristics include the presence of orthodontic appliances or more than one lower anterior tooth with a prosthetic crown or veneer, tumors or significant pathology of the soft or hard tissues of the oral cavity, moderate or advanced periodontal disease, five or more carious lesions requiring immediate care, use of antibiotics or steroids any time during 1 month before entry into the study, participation in any other clinical study or panel test, pregnant or breastfeeding women, history of allergies to dentifrice and personal care ingredients, and allergies to dentifrice products. BODY.MATERIALS AND METHODS.PRETEST PHASE: ORAL SOFT/HARD TISSUE AND CALCULUS EXAMINATION, ORAL PROPHYLAXIS AND TOOTHPASTE USE AT HOME: All subjects received an evaluation of their oral soft/hard tissues by the dental examiner. This examination included an evaluation of the soft and hard palate, gingival mucosa, buccal mucosa, mucosa-gingival fold areas, tongue, sublingual and submandibular areas, salivary glands, and the tonsillar and pharyngeal areas. Candidates were examined by the examining dentist to identify those subjects who demonstrate the presence of supragingival calculus. The scoring procedure used to evaluate calculus formation was the Volpe-Manhold calculus index which uses a standardized and calibrated (in millimeters) periodontal probe to measure supragingival calculus formation in three planes (mesial, midline, distal) of the lingual surfaces of the six lower anterior teeth. Adding the 18 measurements of six teeth constitutes, a total Volpe-Manhold score for each subject.[67] All subjects entered into the pretest phase of the study received a complete oral prophylaxis after the pretest phase calculus examination. The prophylaxis procedure was verified for its thoroughness by use of a red plaque disclosing solution. Subjects were provided a fluoride toothpaste (washout product) for the pretest phase of the study and a soft-bristled adult toothbrush for home use. Subjects were instructed to brush their teeth for 1 min twice daily for 8 weeks. Subjects were instructed to use only the toothpaste provide during the 8-week pretest phase period. There were no restrictions regarding diet and smoking habits during the study. BODY.MATERIALS AND METHODS.TEST PHASE: ORAL SOFT/HARD TISSUE AND CALCULUS EXAMINATION, RANDOMIZATION OF SUBJECTS, ORAL PROPHYLAXIS AND TOOTHPASTE USE AT HOME: After using fluoride toothpaste for 8 weeks, all subjects reported back to the clinical facility. Subjects received an evaluation of their oral soft/hard tissues and examined for calculus formation. The same examination, scoring, and recording procedure employed at the start of the pretest phase of the study were repeated. The same examining dentist performed all examinations. All subjects were randomized into three balanced groups based on their sex and test-phase calculus examination scores. Each group was randomly assigned to one of the three test toothpastes. All toothpastes were packaged in over wrapped tubes so that neither the subjects nor the examining dentist were aware of the product identities during the study. All subjects entered into the test phase of the study received a complete oral prophylaxis after the test phase calculus examination. The prophylaxis procedure was verified for its thoroughness by use of a red plaque disclosing solution. Subjects were provided with their assigned toothpaste for the test phase of the study and a soft-bristled adult toothbrush for home use. Subjects were instructed to brush their teeth for 1 min twice daily. Subjects were instructed to use only their assigned toothpaste during the 12-week test phase period. There were no restrictions regarding diet and smoking habits during the study. BODY.MATERIALS AND METHODS.TEST PHASE: 12-WEEK ORAL SOFT/HARD TISSUE ASSESSMENT AND CALCULUS EXAMINATION: After using their assigned toothpaste for 12 weeks, all subjects reported back to the clinical facility to receive an evaluation of their oral soft/hard tissues and calculus formation. The same examination, scoring, and recording procedure employed at the start of the test phase were repeated. The same examining dentist performed all examinations. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: Statistical analysis was calculated using the statistical program SPSS for window version 18.0 (SPSS Inc., Chicago, IL, USA). All statistical tests of hypotheses used two-way analysis of variance (two-way ANOVA) and employed a level of statistical significance of P < 0.05. BODY.RESULTS: All 150 subjects who entered the study completed this clinical study. The characteristics and test phase Volpe-Manhold calculus index scores of subjects in the three test toothpastes are given in Table 1. The Cavity Protection group consisted of 50 subjects (19 males and 31 females), aged between 20 and 53 years (mean 35.16 years), and had calculus score of 0.79. The Abhaibhubejhr group comprised 50 subjects (22 males, 28 females), aged between 21 and 54 years (mean 36.06 years), and had calculus score of 0.77 while the Total group had 50 subjects (20 males, 30 females), aged 27–51 years (mean 35.80 years), and calculus score of 0.76. An ANOVA indicated no statistically significant difference among the three toothpaste groups (P > 0.05). The three study groups were well-balanced based on the number of subjects, age, gender, and test phase calculus scores. Table 1 Number, age, gender, and test phase calculus scores of subjects in the three study groups Table 2 shows the comparison of the Volpe-Manhold calculus index scores of the three test toothpastes after 12 weeks of assigned toothpaste use. The mean 12-week calculus scores for the Cavity Protection, Abhaibhubejhr, and Total toothpaste groups were 0.78, 0.62, and 0.48, respectively. The subjects who used the Abhaibhubejhr and Total toothpastes for 12 weeks showed 20.51% and 38.46% less calculus formation, respectively than subjects who used the Cavity Protection toothpaste, which was statistically significant (P < 0.05). In addition, subjects using the total toothpaste had 22.58% statistically significant less calculus formation than subjects using the Abhaibhubejhr toothpaste (P < 0.05). Table 2 The mean 12 weeks calculus scores for the three toothpaste groups and % calculus reduction BODY.DISCUSSION: Tooth brushing is relatively effective in dental plaque removal, but it is still inadequate for the maintenance of gingival health. Chemotherapeutic agents have been used to supplement the mechanical removal of dental plaque and calculus from teeth. To the best of our knowledge, this study is the first to evaluate the effect of Abhaibhubejhr Herbal Toothpaste in the control of supragingival calculus formation. This study showed significant anticalculus benefits for the Colgate Total and Abhaibhubejhr toothpastes. Results from this study also showed superior calculus inhibition properties for the Colgate Total versus the Abhaibhubejhr toothpaste. These findings are consistent with other reports in literature showing positive anticalculus benefits for Colgate Total toothpaste.[89] The active ingredients in Colgate Total toothpaste were 0.3% triclosan and 2.0% PVM/MA copolymer, whose mechanism of action is antimicrobial. Triclosan is a broad-spectrum antimicrobial agent active on both Gram-positive and Gram-negative bacteria. At bacterioctatic concentrations, triclosan prevents essential amino acid uptake, while at bactericidal concentrations, triclosan destroys the integrity of the cytoplasmic membrane and causes leakage of cellular contents.[10] The principal ingredients in Abhaibhubejhr Herbal Toothpaste were fruit pericarp of Garcinia mangostana Linn (mangosteen), leaf of Piper betle Linn (betel), and leaf of Psidium guajava Linn (guava). Phytochemical studies showed that mangosteen pericarp contained various bioactive substances including xanthones, tannins, triterpenes, anthocyanins, polysaccharides, phenolic compounds, Vitamin B1, B2, and C.[11] These xanthones exhibit a variety of biological and pharmacological properties including antibacterial, anti-inflammatory, antiviral, antifungal, antioxidant, antiallergy, analgesic, and anticancer.[1213] Due to its antimicrobial activity, mangosteen pericarp could probably lead to the reduction of plaque and calculus formation. Some studies suggested that mouthwash containing mangosteen pericarp has benefits as an adjunct to periodontal treatment and for control of oral malodor.[1415] Betel leaf contains volatile oil (eugenol, cadinene, carvacrol, caryophyllene, chavitbetol, chavicol, estragole, amino acids, pyridine alkaloids, sitosterols, tannins, stigmasterol, Vitamin C, oxalic acid, malic acid, and inorganic elements like fluoride and iron).[16] Further betel leaf possesses anti-bacterial, anti-cariogenic, anti-inflammatory, antifungal, antiprotozoan, anti-allergic, antidiabetic, hepatoprotective, anti-ulcer, cardioprotective, antihyperlipidemic, anti-platelet, and immunomodulatory properties.[17] Guava leaf contains essential oils, flavonoids, tannins, eugenol, saponins, triterpenes, malic acid, and phenolic acid.[18] The potential pharmacologic activities of the extract from guava leaf have been reviewed including antimicrobial, antioxidant, anti-allergy, anti-genotoxic, anti, antiplasmodial, antispasmodic, cardioactive, cough suppressant, antidiabetic, anti-inflammatory, antinociceptive, and antiplaque.[19] It has been reported that the extract of guava leaf reduced the adherence of early plaque bacteria to an experimental pellicle.[20] Razak and Rahim,[21] in 2006, also found that treatment of the early plaque settlers with aqueous guava extract reduced the cell-surface hydrophobicity of Streptococcus sanguinis, Streptococcus mitis, and Actinomyces sp. The extract was observed to have the ability to alter and disturb the surface characteristics of the early plaque settlers and make them less adherent leading to its potential as an antiplaque and anticalculus agent. Recently, there is the study that revealed differences in the antimicrobial activities of commercial natural toothpastes and suggested that the toothpastes with natural compounds have therapeutic potential and need searching in more detail for the clinical applications.[22] In addition, a recent study indicated that a gel preparation containing 10% Lippia sidoides essential oil was an efficient herbal antiplaque and antigingivitis agent.[23] BODY.CONCLUSION: This study evaluated the anti-calculus efficacy of Abhaibhubejhr Herbal Toothpastes compared to a positive control toothpaste (Colgate Total) and a negative control toothpaste (Colgate Cavity Protection). The Abhaibhubejhr and Colgate Total toothpastes revealed significant anti-calculus efficacy compared to the control. The Colgate Total had superior anti-calculus efficacy compared to the Abhaibhubejhr toothpaste. BODY.CONCLUSION.FINANCIAL SUPPORT AND SPONSORSHIP: This study was supported by Mahidol University, Faculty of Dentistry Grant (2013). BODY.CONCLUSION.CONFLICTS OF INTEREST: There are no conflicts of interest.

TITLE: Complications of Lumbar Artificial Disc Replacement Compared to Fusion: Results From the Prospective, Randomized, Multicenter US Food and Drug Administration Investigational Device Exemption Study of the Charité Artificial Disc ABSTRACT.BACKGROUND: Previous reports of lumbar total disc replacement (TDR) have described significant complications. The US Food and Drug Administration (FDA) investigational device exemption (IDE) study of the Charité artificial disc represents the first level I data comparison of TDR to fusion. ABSTRACT.METHODS: In the prospective, randomized, multicenter IDE study, patients were randomized in a 2:1 ratio, with 205 patients in the Charité group and 99 patients in the control group (anterior lumbar interbody fusion [ALIF] with BAK cages). Inclusion criteria included confirmed single-level degenerative disc disease at L4-5 or L5-S1 and failure of nonoperative treatment for at least 6 months. Complications were reported throughout the study. ABSTRACT.RESULTS: The rate of approach-related complications was 9.8% in the investigational group and 10.1% in the control group. The rate of major neurological complications was similar between the 2 groups (investigational = 4.4%, control = 4.0%). There was a higher rate of superficial wound infection in the investigational group but no deep wound infections in either group. Pseudarthrosis occurred in 9.1% of control group patients. The rate of subsidence in the investigational group was 3.4%. The reoperation rate was 5.4% in the investigational group and 9.1% in the control group. ABSTRACT.CONCLUSIONS: The incidence of perioperative and postoperative complications for lumbar TDR was similar to that of ALIF. Vigilance is necessary with respect to patient indications, training, and correct surgical technique to maintain TDR complications at the levels experienced in the IDE study. BODY.INTRODUCTION: Total disc replacement (TDR) has been used to treat lumbar degenerative disc disease for 2 decades with varying success rates. As with any procedure in any surgical discipline, complications can and do occur. The Charité artificial disc (DePuy Spine, Raynham, Mass) was first made commercially available outside the United States in 1987. In October 2004, the device was approved by the US Food and Drug Administration (FDA) for the treatment of lumbar degenerative disc disease (DDD) at 1 level: L4-5 or L5-S1. The literature contains multiple reports of the worldwide experience with lumbar TDR, almost all of which are retrospective case series or case reports that have been described previously in review articles.1–4 Because the Charité artificial disc has the longest history of any TDR device, more complications have been reported in the literature for it than for other TDR devices. In 2003, van Ooij et al.5 reported 27 complications of TDR with the Charité artificial disc. However, what was not reported, as described by McAfee, 6 was that these complications came from a series of 500 patients with widely varying patient indications, the use of early basic instrumentation, and, in some cases, inadequate sizing of the prosthesis. Zeegers et al.7 reported a retrospective series of 50 patients implanted with the Charité artificial disc with a complication rate of 13%. In a series of 46 patients, Cinotti et al.8 reported a reoperation rate of 19.5%. Other authors have reported similar or lower complication rates from TDR with the Charité artificial disc. Lemaire et al.9 reported a total of 21 complications, both major and minor, in their series of 100 patients with minimum 10-year follow-up. David10 reported a 14% complication rate in 197 patients followed for 10 years. All of these series were retrospective, involving the early experience of the authors with lumbar TDR. Since the time of these early implantations with the Charité artificial disc, patient selection criteria, surgical implantation instruments, and surgical technique have all evolved, in part as a response to the early failures reported. To date, an analysis of complications of lumbar TDR, using prospective, narrow, clinically defined indications and a standardized surgical technique with modern instrumentation have not been reported as part of a prospective, randomized, controlled trial. BODY.MATERIALS AND METHODS: Between May 2000 and April 2002, 304 patients underwent surgery in this prospective, randomized, nonblinded, FDAapproved study at 14 investigational sites across the United States. Before beginning patient enrollment, each site obtained local institutional review board approval for conducting the study. Complete inclusion and exclusion criteria were previously described by both Geisler et al.11 and Blumenthal et al.12 The primary inclusion criteria were single-level symptomatic DDD at L4-5 or L5-1 confirmed by provocative discography, back or leg pain without nerve root compression (radiculopathy) with a visual analog scale (VAS) score of ≥40 (range: 1–100) and an Oswestry Disability Index 2.013 score of ≥30 (range: 1–100), ability to tolerate an anterior abdominal approach, and failure to respond to nonoperative treatment for a period of at least 6 months. The primary exclusion criteria were multilevel symptomatic DDD, previous thoracic or lumbar fusion, current or previous lower lumbar fracture, osteoporosis, spondylolisthesis >3 mm, spondylosis, or scoliotic deformity greater than 11°. Patients were randomly assigned in a 2:1 ratio to one of 2 groups. The investigational group received TDR with the Charité artificial disc. The control group received anterior lumbar interbody fusion (ALIF) with BAK threaded fusion cages (Zimmer Spine, Minneapolis, Minn) packed with iliac crest autograft. A total of 205 patients were enrolled in the investigational group, and 99 patients were enrolled in the control group. There was no significant difference between the groups with respect to all demographic variables with the exception of mean weight, which was slightly higher in the control group (Table 1). Table 1 Patient Demographics: Food and Drug Administration Investigational Device Exemption Study of the Charité Artificial Disc Investigational Group (N = 205) Control Group (N = 99) P Gender, no. (%)  Men 113 (55.1) 44 (44.4) .088  Women 92 (44.9) 55 (55.6) Age, y  Mean (SD) 39.6 (8.16) 39.6 (9.07) .946  Median 40.0 39.0  Range 19-60 20-60   > 45, no. (%) 47 (22.9) 30 (30.3) .205   ≤ 45, no. (%) 158 (77.1) 69 (69.7) Race, no. (%)  White 188 (91.7) 87 (87.9) .540  African American 8 (3.9) 5 (5.0)  Other 9 (4.4) 7 (7.1) Height, cm  Mean (SD) 172.3 173.6 .249  Median 170.2 172.7  Range 150-201 155-196 Weight, kg  Mean (SD) 77.5 (15.67) 81.7 (16.46) .035  Median 77.1 79.4  Range 46-120 51-122 Body mass index a  Mean (SD) 26.0 (4.23) 27.0 (4.76) .056  Median 26.0 26.9  Range 17-39 18-10 Previous spinal surgery, no. (%)  Yes 70 (34.1) 33 (33.3) .999  No 135 (65.9) 66 (66.7) Normal activity level before experiencing back pain, no. (%)  Active 188 (91.7) 86 (86.9) .284  Moderate 15 (7.3) 11 (11.1)  Light 1 (0.5) 2 (2.0)  Minimal 1 (0.5) 0 Activity level at enrollment, no. (%)  Active 9 (4.4) 1 (1.0) .064  Moderate 26 (12.7) 5 (5.0)  Light 54 (26.3) 27 (27.3)  Minimal 116 (56.6) 66 (66.7) Preoperative work status, no. (% working) 109 (53.2) 57 (57.6) .470 Note. Fisher's exact test was used to test categorical variables. Student's t test was used to test means. a Weight (in kg) divided by height (in m)2. A detailed surgical technique for implantation of the Charité artificial disc was previously described by Geisler.14 Patients enrolled in the study were implanted with either the investigational device or the control device through an open (non–minimally invasive) anterior retroperitoneal approach. Clinical and radiographic outcomes were previously described by Blumenthal et al.12 and McAfee et al.,15 respectively. The follow-up rate at 24 months within the time window described by the protocol was 91.5% in the investigational group and 89.2% in the control group. Adverse events were recorded throughout the study, both perioperatively and postoperatively, until the last enrolled patient reached the end of the 24-month follow-up period in April 2004. The reporting of adverse events followed adherence to strict FDA regulations concerning adverse event reporting in an investigational device exemption (IDE) study. All patient files and case report forms were monitored by a contract research organization to ensure that all adverse events were captured and reported to the FDA and local institutional review boards. Final adverse event data from the randomized arm of the study was verified as complete and submitted to the FDA in August 2004, before FDA approval of the investigational device in October 2004. Adverse event data were reviewed by the authors. A large number of reported adverse events in an IDE study are often unrelated to the treatment. Examples include reported events such as dermatological events, psychological events, drug allergy, motor vehicle accidents, and coumadin overdoses. Some adverse events reported were secondary to another event, such as fever with infection and leg pain with a neurological event. Some reported adverse events are normal occurrences after spinal surgery, such as incision pain and musculoskeletal spasms. These adverse events, not related to the procedure or the approach, were excluded from this review to create a true account of the perioperative and postoperative complications associated with a TDR procedure in comparison to an ALIF procedure. BODY.STATISTICAL METHODS: Fisher's exact test was used to test categorical variables between the 2 groups. Student's t test was used to test the difference between means. BODY.RESULTS: The total number of subjects with reported adverse events was 155 (75.6%) in the investigational group (205) and 77 (77.8%) in the control group (99). This total includes all adverse events reported, including the ones excluded from this review. Therefore, approximately one quarter of all patients enrolled in the study had no reported adverse events. BODY.RESULTS.APPROACH-RELATED EVENTS: The rate of approach-related complications in all 304 patients was 9.9% (30/304) and was similar between the 2 groups, with 9.8% (20/205) in the investigational group and 10.1% (10/99) in the control group (Table 2). The rate of venous injury in the investigational group (4.4%) was twice that of the rate in the control group (2.0%). The overall rate of retrograde ejaculation in men was 4.1% (6/147), and it was slightly higher in the control group (5.5%, 3/92) compared with the investigational group (3.3%, 3/55). Of these 6 cases of retrograde ejaculation in both groups, 1 resolved spontaneously 17 months after onset, 3 continued at the latest follow-up, and the prognosis for the last 2 cases of retrograde ejaculation is unknown. There were no cases of either arterial thrombosis or deep vein thrombosis in either group. There was no significant difference with respect to incidence of approach-related complications in the TDR group at L4-5 versus L5-S1 (P = .145, Fisher's exact test). Table 2 Approach-Related Complications: Food and Drug Administration Investigational Device Exemption Study of the Charité Artificial Disc Investigational Group Control Group P (N = 205) (N = 99) All approach related, no. (%) 20 (9.8) 10 (10.1) .925 Venous injury, no. (%) 9 (4.4) 2 (2.0) Retrograde ejaculation, no. (%) a 3 (3.3) 3 (5.5) .515 Ileus, no. (%) 2 (1.0) 1 (1.0) Perioperative vein thrombosis, no. (%) 2 (1.0) 0 Clinically significant blood loss >1500 mL, no. (%) 1 (0.5) 2 (2.0) Incisional hernia, no. (%) 1 (0.5) 2 (2.0) Epidural hematoma, no. (%) 1 (0.5) 0 Dural tear, no. (%) 1 (0.5) 0 Deep vein thrombosis, no. (%) 0 0 Arterial thrombosis, no. (%) 0 0 Note . Fisher's exact test was used to test categorical variables. a Of 92 men in the investigational group and 55 men in the control group. BODY.RESULTS.INFECTION: The rate of all infections was higher in the investigational group (12.7%) compared with the control group (8.1%) (Table 3). The rate of superficial wound infection was 3 times higher in the investigational group (6.3%) compared with the control group (2.0%), but there was no significant difference between the groups (P = .103). Deep wound infections requiring irrigation and debridement or intravenous antibiotics did not occur in either group. There were 3 (3.0%) cases of iliac crest donor site infection in the control group. Table 3 Infection Complications: Food and Drug Administration Investigational Device Exemption Study of the Charité Artificial Disc Investigational Group (N = 205) Control Group (N = 99) P All infection complications, no. (%) 26 (12.7) 8 (8.1) .233 Superficial wound with incision site pain, no. (%) 13 (6.3) 2 (2.0) .103 Other non-wound related, no. (%) 5 (2.4) 1 (1.0) Urinary tract infection, no. (%) 5 (2.4) 1 (1.0) Wound swelling, no. (%) 2 (1.0) 0 Pulmonary complication, no. (%) 1 (0.5) 0 Peritonitis, no. (%) 0 1 (1.0) Iliac crest donor site, no. (%) 0 3 (3.0) Note. Fisher's exact test was used to test categorical variables BODY.RESULTS.NEUROLOGICAL EVENTS: As previously reported and as described in detail by Geisler et al., the overall rate of neurological complications was equivalent between the 2 groups (Table 4). Geisler divided the neurological complications into categories of major, minor, and other. The rate of major neurological adverse events, which included burning or dysthetic pain, motor deficit, or nerve root injury, was 4.4% in the investigational group and 4.0% in the control group (P = .887). The rate of minor neurological events, which included numbness, was 9.8% in the investigational group and 8.1% in the control group. Table 4 Neurological Complications: Food and Drug Administration Investigational Device Exemption Study of the Charité Artificial Disc Investigational Group (N = 205) Control Group (N = 99) P All neurological complications, no. (%) 33 (16.1) 17 (17.2) .813 Major, no. (%) 9 (4.4) 4 (4.0) .888  Burning or dysthetic pain 5 (2.4) 3 (3.0)  Motor deficit—index level related 3 (1.5) 1 (1.0)  Nerve root injury 1 (0.5) 0 Minor, no. (%) 20 (9.8) 8 (8.1)  Numbness—index level related 20 (9.8) 7 (7.1)  Numbness lower sacral root distribution 0 1 (1.0) Other, no. (%) 8 (3.9) 8 (8.1)  Numbness—non-index level-related 5 (2.4) 4 (4.0)  Reflex change 2 (1.0) 2 (2.0)  Positive Waddell signs 1 (0.5) 1 (1.0)  Mechanical signs (SLR) 0 1 (1.0) Note. SLR = straight leg raise. Fisher's exact test was used to test categorical variables. BODY.RESULTS.FUSION TREATMENT–RELATED AND PROSTHESIS-RELATED COMPLICATIONS: The rate of pseudarthrosis in the control group was 9.1% (9/99) (Table 5). There were 8 (3.9%) prosthesis-related complications in the investigational group, which included 7 cases of subsidence of the prosthesis endplate into the vertebral endplate (≥2 mm) and 1 cases of implant displacement >3 mm. There were no catastrophic device failures and no cases of osteolysis in the investigational group. A total of 18 (18.2%) patients in the control group reported bone graft donor site pain. All reports of donor site pain arose within 42 days after surgery. Table 5 Fusion Treatment–Related Events, Device-Related Events, and Reoperation Index Level: Food and Drug Administration Investigational Device Exemption Study of the Charité Artificial Disc Investigational Group (N = 205) Control Group (N = 99) P Fusion treatment related, no. (%) 0 27 (27.3)  Nonunion/pseudarthrosis 0 9 (9.1)  Bone graft donor site pain 0 18 (18.2) Prosthesis related, no. (%) 8 (3.9) 1 (1.0) .163  Collapse or subsidence of implant into adjacent vertebrae 7 (3.4) 1 (1.0)  Implant displacement 1 (0.5) 0 Additional surgery index level, no. (%) 11 (5.4) 9 (9.1) .127  Revision 5 (2.4) 0  Reoperation 4 (2.0) 8 (8.1)  Removal 2 (1.0) 1 (1.0) Note. Fisher's exact test was used to test categorical variables. BODY.RESULTS.REOPERATIONS: Of these 9 patients in the control group with a pseudarthrosis, 8 had supplemental transpedicular fixation after the index surgery for the treatment of their pseudarthrosis. One patient in the control group underwent removal of the BAK cages as a result of misplacement, for a total of 9 reoperations (9.1%). The cages were replaced with an alternative ALIF cage/graft composite and concomitant posterior instrumented fusion. A total of 11 (5.4%) patients in the investigational group had a reoperation at the index level (P = .127). Of these, 5 were revisions to a new Charité prosthesis, usually with a smaller footprint; 4 were supplemental posterior fusions with transpedicular fixation; and 2 were removals with conversion to an ALIF and concomitant instrumented posterior fusion (Table 5). BODY.RESULTS.OTHER COMPLICATIONS: Other reported complications are shown in Table 6. Table 6 Other Complications: Food and Drug Administration Investigational Device Exemption Study of the Charité Artificial Disc Investigational Group (N = 205) Control Group (N = 99) All other complications, no. (%) 9 (4.4) 4 (4.0) Adjacent level DDD 2 (1.0) 1 (1.0) HNP adjacent level 2 (1.0) 1 (1.0) Spondylolisthesis 1 (0.5) 1 (1.0) Spinal stenosis 1 (0.5) 0 Annulus ossification 1 (0.5) 0 Calcification resulting in bridging trabecular bone 1 (0.5) 0 Other lumbar degenerative 1 (0.5) 0 Facet joint degeneration 0 1 Death narcotic related 1 (0.5) 0 Note. DDD = degenerative disc disease; HNP = herniated nucleus pulposus. BODY.RESULTS.PAIN: An adverse event related to postoperative pain was reported for 52.7% (108/205) of patients in the investigational group and for 52.5% (52/99) of patients in the control group. Many patients had multiple reports of pain-related adverse events. Individual adverse event reports for back pain totaled 59 (28.8%) in the investigational group and 32 (32.3%) in the control group. For lower extremity pain alone, the rate was 30.7% (63) in the investigational group and 25.3% (25) in the control group. The rate for a report of both back and lower extremity pain at the same time was 11.7% (24) in the investigational group and 14.1% (14) in the control group. BODY.DISCUSSION: A key point that is often lost in the literature is that the majority of complications related to TDR procedures, particularly complications necessitating revision or removal of a prosthesis, are unrelated to a specific device. In addition, the literature contains just a small subsection of the total worldwide experience with lumbar TDR. A wider experience describing complications and revision strategies for the Charité artificial disc and other lumbar prostheses, including important revision approach considerations, has been previously described by McAfee et al.16 This is a significant educational resource on the topic of complications resulting from lumbar TDR procedures. In this study, the rate of perioperative venous injury was twice as high in the investigational group compared with the control group. This is most likely because of issues with retraction of the great vessels at L4-5 to implant the investigational device. The prosthesis must be placed in the exact center of the disc space, but complete vessel retraction to the right at L4-5 is sometimes difficult to achieve, depending on the patient's vascular anatomy. One other possible cause of the venous injuries in the investigational group may have been the use of first-generation instrumentation throughout the randomized phase of the study. For example, the original endplate inserter had a 30° offset, which forced the instrument laterally onto the patient's left abdomen while inserting the prosthesis endplates. This has since been corrected to a 0° offset with the introduction of new instrumentation (Centreline TDR, DePuy Spine, Raynham, Mass). Because the Charité prosthesis incorporates sharp teeth for vertebral fixation, great care must be taken to avoid a vessel laceration when implanting the prosthesis endplates. Of the 11 perioperative venous injuries, only 1 resulted in blood loss of more than 1500 mL. The overall rate of venous injury in all 304 patients enrolled in the study was 3.6%. This is 2.5 times higher than the rate of venous injury reported by Brau et al.17 in 1315 consecutive ALIF procedures (1.4%). However, the patients in the Brau series may have benefited from the ability to place fusion devices or femoral rings anterolaterally when vessel retraction was insufficient. This was not possible in the IDE study. The higher rate of superficial wound infection in the investigational group may be attributable to the investigational group being more active preoperatively (and postoperatively) compared with the control group. As reported in the clinical results article by Blumenthal et al.,12 although the difference between the groups was not significant, the investigational group had a higher rate of active or moderately active patients preoperatively compared with the control group. The rate of retrograde ejaculation in male patients enrolled in the study was 4.1%. This rate was higher than the rate of 0.3% reported by Brau18 in a study of 17 spine surgeons performing 686 ALIF procedures through a minilaparotomy approach. There is no obvious reason for the disparity between the rate experienced in the IDE trial and the experience of Brau. Sasso et al.19 reported a retrograde ejaculation rate of 1.7% in 116 consecutive cases of ALIF via a retroperitoneal approach and a rate of 13.3% in 30 consecutive cases of ALIF using a transperitoneal approach. Sasso et al.19 recommend avoidance of the transperitoneal approach, except in cases of revision at L5-S1, to reduce the incidence of retrograde ejaculation in male patients. Early identification of the sympathetic plexus and avoiding the use of monocautery also will reduce the incidence of retrograde ejaculation in male patients undergoing a retroperitoneal approach to the lumbar spine. As previously described by Geisler et al.,11 the rate of major neurological complications was small in both groups, and the overall rate of neurological adverse events was equivalent between both groups. Therefore, it can be expected that the incidence of neurological complications as a result of TDR with the Charité artificial disc will be no greater than for ALIF procedures. Subsidence occurred in 7 patients enrolled in the investigational group. We believe that these cases were technique related. It is important to perform a complete discectomy before prosthesis insertion. However, as with an ALIF procedure, removal of the bony vertebral endplate in the central part of the vertebral body will result in exposure of soft cancellous bone, which is unable to biomechanically support the prosthesis under normal loading, causing subsidence. We believe that the discectomy may have been too aggressive in these 7 cases, and we caution against removing the bony endplate during the discectomy phase of the procedure. Further, it is important to size the prosthesis correctly and maximize the surface area so that the footprint of the device covers as much of the vertebral rim of cortical bone as possible. Since FDA approval, 2 additional footprint sizes have been added to assist in matching up the footprint size to the patient's vertebral anatomy. The rate of confirmed nonunion in the control group (9%) further validates the study performed by Kuslich et al.20 using BAK cages for standalone interbody fusion in narrowly indicated patients. In the 7 cases of revision to a new prosthesis (5) or removal and replacement with interbody fusion (2), all of the devices were placed too anteriorly, which led to migration or displacement of the prosthesis. Placement of the prosthesis too anteriorly can lead to migration or core displacement because adequate posterior disc space height is not achieved. Therefore, it is imperative that the prosthesis be placed in the ideal position in the sagittal plane, 2 mm dorsal to the exact center of the disc space, with particular attention to fiuoroscopic landmarks to avoid parallax-related positioning errors. Revisions and reoperations in the randomized arm of the study, as well as the training and continued access arms, are described in greater detail by McAfee et al.21 It is unclear whether many of the complications classified as "other" were a result of the surgical intervention, or whether they were not realized preoperatively. This may be the case for reported events such as postoperative spondylolisthesis and spinal stenosis in both groups. As described by McAfee et al.,21 2 pars fractures in the investigational group were evident retrospectively on preoperative radiographs that were not diagnosed until after the index surgery. Inclusion of these patients in the study was a protocol deviation, and because the fractures were present preoperatively and not caused by the device or the procedure, they are not classified as adverse events or complications. The rate of adjacent-level disc disease is exceedingly low out to 24 months. However, we believe this topic needs to be studied with long-term data because adjacentlevel disc disease does not normally present itself in such a short timeframe. It is unknown how many reports of postoperative pain are true complications in this study, related to either the device or procedure in either group. The protocol did not specify the reporting threshold for postoperative pain. General guidelines to the investigative sites recommended that pain be reported as an adverse event if the pain is (1) (a) possibly, (b) probably, or (c) definitely related to the device or the intervention; (2) more severe than the patient experienced before the intervention; or (3) represented in a new area where the patient did not have pain before the intervention. These guidelines were not adequately communicated to the investigators in the study, so it is not known how many of these adverse event reports fit the definition described previously mentioned. Pain after lumbar surgery is often expected. With respect to point 1a above, almost all patients experience pain related to the procedure in the immediate postoperative timeframe and for several weeks after surgery. At 24 months of follow-up, the mean VAS score was 31.2 in the investigational group and 37.5 in the control group. This represents a significant improvement in pain compared with baseline levels, but clearly some patients in both groups were not pain free. Is postoperative pain a complication of a lumbar TDR procedure or a lumbar fusion procedure? If so, when? In terms of classifying pain as a complication, we should be more concerned with instances of pain fitting the definition of points 2 and 3; pain more severe than before the intervention, and pain in a new area. Perhaps point 2 should be expanded to include the same amount of pain as before the intervention, because certainly "breaking even" on a patient's pain is not a good result for the patient. Unfortunately, because of the reporting of pain-related adverse events in this study, it is not possible to adequately address the true rate of pain-related complications. Complications occur with every surgical procedure in every surgical discipline. However, specific steps can be taken to minimize perioperative and postoperative surgical complications, particularly with respect to lumbar TDR procedures. The first of these steps is correct patient indications. Clinical and radiographic results previously described, as well as the complications reported here, are directly related to the narrow patient indications used in this IDE study. Use of the Charité artificial disc, or any spine surgery device, in nonindicated patients may not yield the same results and may lead to unintended, avoidable complications. Both training and experience are important in terms of minimizing complications. Each of the investigators in this IDE study had extensive experience with ALIF procedures and the retroperitoneal approach to the lumbar spine before enrolling patients in the study. It is imperative that surgeons wishing to add lumbar TDR procedures to their armamentarium attend a company-sponsored training course on the procedure and perform the procedure without shortcuts. This holds true for other TDR prostheses as well, because each prosthesis has its own surgical technique and instrumentation. It is recommended that after training, surgeons observe at least 1 case in the operating room before attempting the procedure. Further, for those surgeons who have performed few or no ALIF procedures, it is recommended that they first become comfortable with ALIF before including TDR procedures in their practice. The senior author prefers to perform his own approaches. However, the coauthors believe an approach surgeon is a valuable addition to the surgical team, to assist with mitigating perioperative complications, as well as for prescreening patients who have had previous abdominal surgery for potential problems during the approach. In our opinion, major neurological complications may be minimized by taking care not to overdistract the disc space. The goal of TDR (as with interbody fusion) is to restore the original disc height relative to the heights of adjacent discs. The goal is not to increase disc height to the maximum possible. TDR procedures differ from interbody fusion procedures because exact placement of the prosthesis is required for the best results. This is not necessarily true for all interbody fusion procedures. Particular attention should be paid to device placement in the ideal position, as described by McAfee et al.15: 2 mm dorsal to the midpoint of the disc space in the sagittal plane and in the exact center in the coronal plane. If the prosthesis is placed too anteriorly, migration or displacement are more likely to occur. If initial placement of the prosthesis is too anterior on fiuoroscopy, we recommend repositioning of the prosthesis before closing the wound. Revision anterior lumbar surgery is often difficult because of scarring in between and around the vessels; therefore, repositioning the prosthesis during the index surgery may eliminate the need for a potentially life-threatening revision surgery in the future. Incidence of perioperative and postoperative complications for lumbar TDR was similar that of ALIF. Vigilance is necessary with respect to patient indications, training, and correct surgical technique to maintain TDR complications at the levels experienced in the IDE study.

TITLE: Comparison of Local Anesthetic Effect of Bupivacaine versus Bupivacaine plus Dexamethasone in Nasal Surgery ABSTRACT.INTRODUCTION:: Adequate pain control is an important consideration in the post-surgical management of patients. Local nerve blockade added to general anesthesia can provide excellent pain control during and after most nasal surgical procedures. The aim of this study was to determine the combined effect of local anesthetic drugs with corticosteroids in nasal surgery. ABSTRACT.MATERIALS AND METHODS:: In this double-blind clinical study, 60 patients who underwent different nasal surgical procedures were matched and divided into two equal groups. Bilateral local nerve blockade was used in both groups. Bupivacaine or bupivacaine plus dexamethasone was administered by injection (groups B and B+D, respectively). Postoperative visual analog scale (VAS) pain values and the need for oral/intramuscular analgesic treatment in the first 24 h were recorded in all patients. ABSTRACT.RESULTS:: Thirty-eight male (63.3%) and 22 female (36.7%) patients were included in this study, with a mean age of 28.3 ± 8.2 years. At 1, 2, 4, 6, and 12 h post surgery, VAS pain values were significantly lower in the B+D group than in the B group. The analgesic requirement was significantly lower in the B+D group compared with the B group. No relevant complications were seen during surgery or postoperative hospitalization. BODY.CONCLUSION: : This study demonstrates the positive effect of a combination of a dexamethasone with a bupivacaine in reducing pain and the need for analgesic drugs after different nasal surgeries. No acute or short-term post-surgical complications were observed in this study. BODY.INTRODUCTION: Appropriate pain control is an important consideration in the post-surgical management of patients. Physicians should ensure that therapeutic procedures are not the cause of extra and unnecessary pain to patients. Pain is particularly common after nasal surgery, especially when bone manipulation and periosteal irritation are involved. Substantial research and clinical observations suggest that pain reduction can be achieved in nasal surgery through use of local anesthesia(1).The use of local anesthesia in combination with general anesthesia is increasingly observed in head and neck surgery. In particular, local nerve blockade added to general anesthesia can provide excellent pain control during and after surgery in most nasal procedures (2,3). Lidocaine and bupivacaine are commonly used for local injection or nerve blockade in nasal surgeries (4). Numerous studies performed in Iran and other countries have demonstrated an advantage of topical administration, local injection, and nerve blockade with bupivacaine or lidocaine versus saline. Other studies have shown an advantage of bupivacaine over lidocaine with regard to pain control and duration of pain control. In this paper, we investigate the combined effect of dexamethasone with bupivacaine as local anesthesia after nasal surgery, in order to determine the optimal procedure for pain control and reduction of analgesic use post surgery. BODY.MATERIALS AND METHODS: This double-blind clinical study was carried out in the otorhinolaryngology ward of Imam-Khomeini Hospital, Ahwaz. A total of 60 patients who underwent different nasal surgical procedures (including closed nasal bone reduction, septoplasty, rhinoplasty and functional endoscopic sinus surgery (FESS)) under general anesthesia were matched according to demographic factors such as age, sex, BMI, type of surgery,...and divided into two groups of 30. Bilateral local nerve blockade of the infraorbital nerve, supratrochlear nerve, and terminal branches of the nasopalatine nerve was used in both groups immediately after general anesthesia. Bupivacaine (Marcaine®; 0.5%, 5–20 cm3, with a mean dose of 1–1.5 mg/kg and a maximum dose of 100 mg) and bupivacaine plus dexamethasone (0.4%, 0.5–2 cm3, with a mean dose of 0.1 mg/kg) was administered by injection to the B and B+D groups, respectively. Visual analog scale (VAS) pain scores (0–100) were recorded 1, 2, 4, 6, 12, and 24 h postoperatively in all patients. A 325mg Acetaminophen (Paracetamol) tablet was available upon patient request postoperatively, with intramuscular Pethidine (Ampul 20mg) as ''rescue'' analgesia. The need for analgesic agents, including oral treatment or intramuscular (IM) injection in the first 24 h after the operation was recorded in a double-blinded manner. Post surgical pain and need to analgesics in the first 24 hours was Calculated and compared in any type of nasal surgery separately,and then final results were gathered together and Analysised Generally. T-tests and chi-square tests were used for comparison of quantitative and qualitative variables. Final data were analyzed with descriptive statistics using with SPSS software (16th Edition). BODY.RESULTS: Thirty-eight male (63.3%) and 22 female (36.7%) patients were included in this study, with a mean age of 28.3 ± 8.2 years. Patients underwent closed nasal bone reduction, septoplasty, FESS, and/or rhinoplasty (Fig 1). There were no significant differences between the groups with respect to age, gender, BMI, duration of operation, or type of surgery (Table 1). At 1, 2, 4, 6, and 12 h post surgery, VAS pain scores were significantly lower in the B+D group than in the B group(P<0.0001, P=0.002, P=0.023, P<0.0001 and P=0.011, respectively) but there was no statistically significant difference between the groups in VAS at 24 h after surgery P=0.221 (Table 2). Fig 1Different types of performed nasal surgery in B and B+D groups Table 1 Patients’demographic data Group B+D (n=30) Group B (n=30) Age (Years) 29.8±7.3 26.8±9.1 Sex (M/F) (20/10) (18/12) BMI (kg/cm2) 25.2±3.8 24.7±3.9mk Main surgical duration (Min) 92.3±19 83.4±23 Table 2 Visual Analog Scale (VAS) pain scores at 1, 2, 4, 6, 12, and 24 h post surgery in 60 patients VAS Scores Group B+D(n=30) Group B (n=30) P 1 h post surgery 18.7±2.3 40.9±3.9 <0.0001 2 h post surgery 22.3±1.9 35.1±3.1 0.002 4 h post surgery 18.9±2.4 31.2±2.9 0.023 6 h post surgery 5.7±1.2 16.6±1.9 <0.0001 12 h post surgery 11.2±1.7 21.9±2.4 0.011 24 h post surgery 8.3±1.6 6.2±1.3 0.221 The analgesic requirement was significantly lower in the B+D group compared with the B group (P=0.038), with 12 and 18 patients requiring post-surgical oral/intra muscular analgesics, respectively (Table 3). No relevant complications (such as reduced blood pressure, bradycardia, visual problems, or hypersensitivity reactions) were observed during surgery or in the first 24 h of postoperative hospitalization. Table3 Requirement for post-surgical analgesics in 60 patients Analgesic consumption Group B+D (n=30) Group B (n=30) Nil additional Analgesic 18 12 Oral Analgesic requirement 9 13 1 tablet (Acetaminophen 325 mg) 5 4 2 tablet (acetaminophen 325 mg) 2 4 3 tablet (acetaminophen 325 mg) 1 3 4 tablet (acetaminophen 325 mg) 1 2 Oral and intramuscular (20 mg pethidine) analgesic 3 5 BODY.DISCUSSION: Local anesthetic injection and nerve blockade have been used for many years, but the techniques are now more popular than ever. The major advantages of these techniques, whether used alone or adjacent to general anesthesia are their inherent simplicity and safety. Recently, studies have shown that local injection with bupivacaine (≤0.25% density) causes in local anesthesia and vasoconstriction (5). The effect of different local anesthetic agents in comparison with normal saline and each other was reviewed in various studies. One study done by Edward and Colleagues in university of California demonstrated beneficial effect of bupivacaine versus normal saline in pain control after nasal surgery (6). This advantage for using of bupivacaine versus normal saline was proved in many other studies (7-9). Advantage of local injection with bupivacaine 0.25% in comparison with lidocaine 2%+ Epinephrine 1/100,000 in pain control and reduction need for post-surgical analgesics was shown In another study done by Yavuz and Colleagues in 2008 (10). This Finding also was seen in Yilmaz and Colleagues' study (11). In this investigation, no study was seen relative to combination corticosteroids with local anesthetic agents in Otorhin- olaryngologic fields but using this technics in other body areas was reported, as in study done by Kopacz and Colleagues in university of Seattle adding dexamethasone to bupivacaine for intercostal nerve blockade reduced time for local anesthetic's onset and prolonged painless and senseless duration significantly (12). In another study was done in university of Tehran in 2005, Doctor Movafegh and his cooperators pointed to positive effect of adding dexamethasone to lidocaine in lengthen duration of Brachial plexus nerve blockade (13). In our study, we observed a statistically significant benefit for the combination of bupivacaine and dexamethasone in terms of pain control and a reduced need for analgesic agents in the first 12 h after different nasal surgeries. BODY.CONCLUSION: The results from the present study demonstrate a positive benefit for the combination of dexamethasone with bupivacaine in the reduction of pain and requirement for analgesic drugs after different nasal surgical procedures. No acute or short-term post-surgical complications were observed in our study. Further larger studies seems to be inevitable in order to fully investigate these positive benefits.

TITLE: Urogenital function in robotic vs laparoscopic rectal cancer surgery: a comparative study ABSTRACT.PURPOSE: Urological and sexual dysfunction are recognised risks of rectal cancer surgery; however, there is limited evidence regarding urogenital function comparing robotic to laparoscopic techniques. The aim of this study was to assess the urogenital functional outcomes of patients undergoing laparoscopic and robotic rectal cancer surgery. ABSTRACT.METHODS: Urological and sexual functions were assessed using gender-specific validated standardised questionnaires. Questionnaires were sent a minimum of 6 months after surgery, and patients were asked to report their urogenital function pre- and post-operatively, allowing changes in urogenital function to be identified. Questionnaires were sent to 158 patients (89 laparoscopy, 69 robotic) of whom 126 (80 %) responded. Seventy-eight (49 male, 29 female) of the responders underwent laparoscopic and 48 (35 male, 13 female) robotic surgery. ABSTRACT.RESULTS: Male patients in the robotic group deteriorated less across all components of sexual function and in five components of urological function. Composite male urological and sexual function score changes from baseline were better in the robotic cohort (p < 0.001). In females, there was no difference between the two groups in any of the components of urological or sexual function. However, composite female urological function score change from baseline was better in the robotic group (p = 0.003). ABSTRACT.CONCLUSION: Robotic rectal cancer surgery might offer better post-operative urological and sexual outcomes compared to laparoscopic surgery in male patients and better urological outcomes in females. Larger scale, prospective randomised control studies including urodynamic assessment of urogenital function are required to validate these results. BODY.INTRODUCTION: Rectal cancer surgery is associated with a high risk of urological and sexual dysfunction which significantly affects the quality of life of its survivors [1–6]. Although urogenital dysfunction is thought to be multifactorial in nature, intra-operative damage to the autonomic pelvic nerves is considered to be the primary cause [5–7]. It is argued that better visualisation of the pelvic autonomic nerves such as obtained during minimally invasive surgery could enable the preservation of these structures and therefore reduce the incidence of urogenital dysfunction [8]. However, despite laparoscopic total mesorectal excision (TME) becoming the standard approach in much of the modern world, it is still debated whether it has helped improve urogenital dysfunction. In fact, the evidence comparing the urogenital outcomes of open and laparoscopic TME is conflicting, with some studies advocating favourable urogenital outcomes for laparoscopic TME [9] whilst others for open TME [10]. A recently published systematic review concluded that neither approach is superior in terms of urogenital preservation [11]. A probable explanation for this is that laparoscopic rectal surgery is technically difficult [12]. Existing laparoscopic instruments have a restricted range of movement compared with those of the surgeons' hand and are difficult to use in confined spaces such as the pelvis [13, 14]. Robotic surgical systems were introduced to overcome the technical limitations of laparoscopic surgery [15]. With superior three-dimensional views, tremor filtering and angulated instruments, robotic surgery enables precise dissection in narrow surgical spaces such as the pelvis therefore enabling better preservation of fine structures such as the autonomic nerves [13, 16]. Currently, there are only a few studies investigating the urological and sexual outcomes of robotic surgery against those of laparoscopic surgery and these tend to be predominant about male patients. The aim of this retrospective clinical study is to compare the urological and sexual functional outcomes of robotic and laparoscopic rectal cancer surgery using a validated functional questionnaire in both men and women in a high-volume minimally invasive colorectal unit. Only one similar study has been identified and deemed up to date but its sample size is significantly smaller [17]; therefore, our study aims to build upon that evidence base. BODY.METHODS: All patients who underwent potentially curative elective laparoscopic or robotic rectal cancer resections from December 2006 to September 2009 for the laparoscopic group and from May 2013 and September 2014 for the robotic group were identified from a prospectively maintained database. Rectal cancer was defined as cancer present within 15 cm from the anal verge. Surgery was performed in both groups by two surgeons with vast laparoscopic and robotic experience working in a high-volume minimally invasive colorectal unit. Urological and sexual function was assessed using gender-specific validated questionnaires sent a minimum of 6 months after surgery to allow for wound healing and objective data collection. Urogenital data for the laparoscopic group was collected during a previous study comparing the urogenital outcomes of laparoscopic and open rectal surgery [9]. Urogenital data for the robotic group was collected subsequently, when the two performing surgeons adopted its practice. Formal oncological and physical assessment was undertaken by all patients prior to surgery following the protocol. Pre-operative staging was performed by colonoscopy or CT colonography, computed tomography (CT) of the chest and abdomen and magnetic resonance imaging (MRI) of the pelvis. Patients with low rectal cancers (5 cm from anal verge) underwent additional staging with endo-anal ultrasound (EUS). All patient findings were discussed in the multidisciplinary team meeting prior to initiating any type of treatment. In general, pre-operative long-course chemoradiotherapy was reserved for T4 rectal cancers or those where the circumferential resection margin (CRM) appeared threatened on MRI, whilst short-course radiotherapy was advised for rectal cancers that approached but did not threaten the CRM. Radiotherapy was not used where rectal cancers were considered resectable by TME with a good likelihood of clear margins. Appropriate approval for the study was obtained by the Research and Development department of Portsmouth NHS Trust. Informed consent was also obtained from the patients participating in this study. BODY.METHODS.SURGICAL TECHNIQUE: The laparoscopic group had a standardised technique which has been previously published [9, 18]. A modular approach of medial to lateral colonic mobilisation with isolation and ligation of the main vessels using clips was applied, and TME was performed using monopolar diathermy as previously described [18]. Robotic rectal resections were performed using a single docking fully robotic approach [19]. The principle of standardised technique developed for laparoscopic surgery was also used for robotic surgery. Procedures commenced with medial to lateral dissection followed by vascular control by ligating the main vessels. Hem-o-loks® were used to secure the vessels before division and a three-step approach was used for splenic mobilisation [20]. TME was performed in the same stepwise manner as in the laparoscopic group, starting with posterior mobilisation followed by right lateral, anterior and left lateral mobilisation in a stepwise manner. Similarly, robotic dissection was performed using monopolar diathermy. Post-operatively, all patients were managed using the enhanced recovery program described by Kehlet and Wilmore [21]. Patients were discharged home according to set criteria for discharge. BODY.METHODS.PATIENT SELECTION: No specific selection criteria were used to allocate patients to laparoscopic or robotic surgery. In this study, two surgeons performed all laparoscopic and robotic resections. Between 2006 and 2009, laparoscopy was the preferred approach for rectal surgery, whereas following the adoption of robotic surgery in May 2013, robotic surgery became the surgical approach of choice for rectal cancers. BODY.METHODS.UROGENITAL FUNCTION ASSESSMENT: Urogenital function was assessed as described in our previous study [9]. Anonymous and confidential questionnaires were sent to all surviving patients in February 2010 for the laparoscopic group and in May 2015 for the robotic group, a minimum of 6 months following surgery through the post. Patients were asked to rate their urological and sexual function pre-operatively and post-operatively. To maximise patient response rate, patients that did not reply to the questionnaires within 4 weeks were sent a reminder letter. For male urological function, we used a modification of the International Prostatic Symptoms Score (IPSS) [22], for male sexual function the International Index of Erectile Function (IIEF-5) [23], for female urological function the King's Health questionnaire [24] and for female sexual function the Female Sexual Function Index (FSFI) [25]. The following components were assessed in each questionnaire:Male urological function (MUF): frequency, nocturia, urgency, straining, poor flow and incomplete bladder emptying.Male sexual function (MSF): libido, erection, stiffness for penetration and orgasm/ ejaculation.Female urological function (FUF): frequency, nocturia, urgency and stress incontinence.Female sexual function (FSF): arousal/ libido, lubrication, orgasm and dyspareunia. Overall, there were six components for MUF and four for MSF, FUF and FSF. Each component was analysed independently and a composite score for each questionnaire was created by adding the scores of each component. Scoring was standardised and quantified for all questions. The following scale was applied: 0 for not at all, 1 for less than half the time, 2 for about half the time, 3 for more than half the time and 4 for almost always. A score of 0 reflected normal function whilst 4 poor function. Overall, a high score reflected a high degree of dysfunction whereas a low score reflected normal function. BODY.METHODS.STATISTICAL ANALYSIS: IBM SPSS version 22 (SPSS Inc., Chicago, IL, USA) and Microsoft Excel 2010TM were used for the statistical analysis. Data was expressed as mean ± standard error of the mean and median with interquartile range for parametric and non-parametric data, respectively. Baseline demographic and clinical characteristics were compared using the χ 2 test or Fisher's exact test for categorical variables and the t test or Mann-Whitney U test for continuous variables. Urogenital functional scores were compared using a t test. p values of <0.05 were considered statistically significant. Sexually inactive patients were excluded from the sexual outcome analysis to avoid skewing of the data. BODY.RESULTS.PATIENT CHARACTERISTICS: Questionnaires were sent to 158 patients (89 laparoscopic group, 69 robotic) of whom 126 (80 %) responded. Seventy-eight (49 male, 29 female) of the responders underwent laparoscopic and 48 (35 male, 13 female) robotic surgery. Of those, 45 patients (36 male, 9 female) were sexually active in the laparoscopic group and 17 (13 male, 4 female) in the robotic group. The demographic, clinical and pathological characteristics of the patients included in this study are summarised in Table 1. The baseline characteristics of the two groups were broadly comparable. Nevertheless, patients in the robotic group had lower rectal cancers (p = 0.032) and were more likely to receive long-course pre-operative radiotherapy (p = 0.012) and neoadjuvant chemotherapy (p = 0.030).Table 1Baseline demographic and clinico-pathological featuresLaparoscopicRobotic p valueGender •Male49350.243 •Female2913Age Median (IQR)70 (63–75.25)69 (64–74.75)0.966BMI median (IQR) •Male26 (23–29.5)27 (25–28.25)0.275 •Female26 (24–32.5)27 (24.25–29.5)1.000ASA grade •112 (15 %)4 (9 %)0.407 •251 (65 %)39 (85 %) 0.019 •315 (19 %)3 (7 %)0.066Type of surgery ❖Total AR63 (81 %)40 (84 %)0.716 •Male AR38 (76 %)30 (86 %)0.409 •Female AR25 (86 %)10 (77 %)0.657 ❖Covering ileostomy56 (89 %)34 (85 %)0.562 •Male36 (95 %)26 (87 %)0.394 •Female20 (80 %)8 (80 %)1.000 ❖Total APER14 (18 %)7 (15 %)0.623 •Male10 (20 %)4 (11 %)0.377 •Female4 (14 %)3 (23 %)0.657 ❖ Total Hartman's1 (1 %)1 (2 %)1.000 •Male1 (2 %)1 (3 %)1.000 •Female001.000Anal verge mean (SE)9.36 ± 0.387.91 ± 0.54 0.032 T stage •Tx4 (5 %)2 (4 %)1.000 •T16 (8 %)6 (13 %)0.372 •T226 (33 %)21 (44 %)0.240 •T336 (46 %)16 (33 %)0.156 •T46 (8 %)3 (6 %)1.000Radiotherapy •Pre-op short9 (12 %)0 0.014 •Pre-op long5 (6 %)11 (23 %) 0.012 •Pre-op total14 (18 %)11 (23 %)0.497 •Post-operative01 (2 %)0.328Chemotherapy •Neoadjuvant7 (9 %)11 (23 %) 0.030 •Adjuvant18 (23 %)16 (33 %)0.208Post-op complications •Anastomotic leak3 (4 %)4 (5 %)1 (2 %) •Return to theatre01.0000.301 BODY.RESULTS.MALE UROLOGICAL FUNCTION: There were 49 patients in the laparoscopic group and 35 in the robotic group. In Tables 2 and 3 and Fig. 1, we present the mean pre-operative MUF scores and their change from baseline. Pre-operative urological function was worse across three components (frequency, nocturia, urgency) in the robotic group, and the pre-operative composite mean MUF score was worse in the robotic group.Table 2Baseline and change from baseline MUF mean scores (mean ± standard error of the mean)LaparoscopicRobotic p valueBaseline MUF •Frequency1.632.51 0.013 •Nocturia2.062.91 0.013 •Urgency0.591.63 0.003 •Initiation/straining0.160.260.576 •Poor flow0.691.260.090 •Incomplete bladder emptying0.921.200.406Change from baseline •Frequency0.57 ± 0.16−0.31 ± 0.22 0.002 •Nocturia0.63 ± 0.17−0.20 ± 0.19 0.002 •Urgency0.69 ± 0.21−0.66 ± 0.29 <0.001 •Initiation/straining0.39 ± 0.120.09 ± 0.130.094 •Poor flow0.73 ± 0.18−0.14 ± 0.21 0.002 •Incomplete bladder emptying0.16 ± 0.20−0.63 ± 0.26 0.017 Table 3Mean composite MUF scoresPre-opPost-op p valueMean score changeLap6.069.24 <0.001 3.18 ± 0.69Robotic9.777.69 0.023 −2.14 ± 0.87 p value 0.003 0.229 <0.001 Fig. 1Change in mean composite scores from baseline Mean score change from baseline was better in all except one component (initiation/ straining) in the robotic group (see Table 2). Composite mean MUF score change from baseline was also better in the robotic group as illustrated in Table 3 and Fig. 1. Overall, composite mean MUF scores deteriorated in the laparoscopic group and improved in the robotic group (p < 0.001, p = 0.023), Table 3. BODY.RESULTS.MALE SEXUAL FUNCTION (MSF): There were 36 patients (73 %) in the laparoscopic group and 13 (37 %) in the robotic group who were sexually active. Pre-operative scores were similar in both groups. In Tables 4 and 5 and Fig. 1, we present the mean pre-operative MSF scores and their change from baseline.Table 4Baseline and change from baseline MSF mean scores (mean ± standard error of the mean)LaparoscopicRobotic p valueBaseline MSF •Sexually activeYes 36, no 13Yes 13, no 22 •Libido/arousal0.310.540.422 •Erection0.690.850.712 •Stiffness for penetration0.861.150.547 •Orgasm/ejaculation0.170.920.057Change from baseline •Libido/arousal1.56 ± 0.280 ± 0.30 0.001 •Erection1.53 ± 0.290 ± 0.20 <0.001 •Stiffness for penetration1.39 ± 0.29−0.38 ± 0.21 <0.001 •Orgasm/ejaculation1.78 ± 0.31−0.15 ± 0.25 <0.001 Table 5Mean composite MSF scoresPre-opPost-op p valueMean score changeLap2.318.56 <0.001 6.25 ± 1.08Robotic3.622.920.759−0.69 ± 0.85 p value0.319 0.004 <0.001 Mean scores deteriorated across all component of MSF in the laparoscopic group but none in the robotic group. Statistical comparison of the mean change of MSF scores from baseline revealed favourable outcomes for the robotic group across all four components of MSF as shown in Table 4. Composite MSF score change was also better in the robotic group as illustrated in Table 5 and Fig. 1. BODY.RESULTS.FEMALE UROLOGICAL FUNCTION: There were 29 patients in the laparoscopic group and 13 in the robotic group. The mean pre-operative FUF scores of both groups are shown in Tables 6 and 7. Pre-operative scores were similar in both groups across all components.Table 6Baseline and change from baseline FUF mean scores (mean ± standard error of the mean)LaparoscopicRobotic p valueBaseline FUF •Frequency1.662.230.325 •Nocturia1.792.850.056 •Urgency0.761.460.190 •Stress incontinence1.101.920.143Change from baseline •Frequency0.62 ± 0.25−0.54 ± 0.560.077 •Nocturia0.69 ± 0.270.38 ± 0.400.533 •Urgency0.48 ± 0.16−0.15 ± 0.270.059 •Stress incontinence0.10 ± 0.11−0.23 ± 0.280.287 Table 7Mean composite FUF scoresPre-opPost-op p valueMean score changeLap5.317.21 <0.001 1.90 ± 0.50Robotic8.466.230.065−2.23 ± 1.10 p value0.0520.501 0.003 The mean FUF score changes from baseline are outlined in Tables 6 and 7 and illustrated in Fig. 1. There was no statistical difference in any of the mean FUF component score change from baseline between the two groups. However, composite mean FUF score change from baseline was better in the robotic group. Composite mean FUF score deteriorated in the laparoscopic group but not in the robotic group (see Table 7). BODY.RESULTS.FEMALE SEXUAL FUNCTION: There were nine (31 %) sexually active patients in the laparoscopic group and four (31 %) in the robotic group. Their pre-operative scores were similar in both groups as shown in Tables 8 and 9.Table 8Baseline and change from baseline FSF mean scores (mean ± standard error of the mean)LaparoscopicRobotic p valueBaseline FSF •Sexually activeYes 9, no 20Yes 4, no 9 •Arousal/ libido0.891.500.589 •Lubrication1.441.250.865 •Orgasm0.442.250.177 •Dyspareunia0.440.750.620Change from baseline •Arousal/ libido0.67 ± 0.37−0.25 ± 0.250.066 •Lubrication0 ± 0.171.25 ± 0.950.279 •Orgasm0.89 ± 0.420 ± 00.069 •Dyspareunia0.44 ± 0.472 ± 0.820.159 Table 9Mean composite FSF scoresPre-opPost-op p valueMean score changeLap3.225.220.1542 ± 1.27Robotic5.758.750.1813 ± 1.73 p value0.2510.2690.657 Mean FSF score changes from baseline are outlined in Tables 8 and 9 and Fig. 1. Overall, there was no statistical difference between the mean change of scores from baseline between the two groups in any of the FSF components or the FSF composite scores. BODY.DISCUSSION: Robotic rectal surgery has been gaining popularity over the last few years. However, whether it is superior to laparoscopic rectal surgery remains an open debate. In this study, we have found that the robotic approach offers favourable post-operative urogenital functional outcomes in men and urological outcomes in women. Whilst composite MUF, MSF and FUF scores deteriorated in the laparoscopic group, this was not the case for the robotic group. Mean composite score change from baseline for MUF, MSF and FUF favoured the robotic group. The functional score change from baseline was also statistically better in all four components of MSF and in five out of six components in MUF. Favourable post-operative male sexual function for robotic TME as compared to laparoscopic TME has been demonstrated in previous studies [17, 26–29]. Park et al's [26] study showed that MSF recovers faster in the robotic group (6 vs 12 months), and at 6 months, the overall MSF scores showed a significantly smaller decrease from baseline in the robotic group (p = 0.03). Kim et al. [27], Park et al. [28], D'Annibale et al. [29] and Morelli et al. [17] all demonstrated favourable male sexual outcomes for the robotic group but unlike ours and Park et al's [26] studies, failed to demonstrate a change of overall MSF scores from baseline in favour of the robotic group. It is worth noting that regarding sexual function, the sample size of these studies was similarly small as to our study, with the range of patients varying between 20 and 14 in the robotic group and 23 and 15 in the laparoscopic group. This shows that a relatively small sample size is sufficient to demonstrate the superiority of robotic rectal surgery in terms of male post-operative sexual function outcomes. Unlike sexual function, advantages in male urological function following robotic surgery have been harder to demonstrate. A multitude of studies comparing the MUF of robotic vs laparoscopic patients showed no difference in their outcomes [17, 28, 29]. In contrast to the above, Cho et al. [30] in a retrospective study of 278 patients in each group found that at 1 month after surgery, the voiding dysfunction rate was higher in the laparoscopic group (4.3 vs 0.7 %, p = 0.012). However, this study did not apply any functional scores for the assessment of dysfunction, leaving it open to observation bias. Park et al. [26] on a population of 32 patients in each group found that at 12 months following surgery, MUF score change from baseline was lower in the robotic group, but this was not quite statistically significant (p = 0.051). However, Kim et al. reported a clear advantage for robotic TME in terms of urological function [27]. On a sample size of 30 robotic and 39 laparoscopic patients, they showed that urological function recovered faster in the robotic group (3 vs 6 months) and functional score change from baseline was lower in the robotic group at 3 months (p = 0.036). However, we should note that for this study, male and female data were combined. Our study is the first one to demonstrate a significant favourable overall score change from baseline for the robotic approach in a male cohort only. This is probably due to our study's larger sample size (49 laparoscopic vs 35 robotic). It is also worth noting that the composite MUF mean score improved in the robotic group. Obviously, we are not suggesting that robotic rectal surgery might itself improve MUF. We believe this might have occurred due to a number of factors. First of all, our study was open to recall bias, since patients reported their urogenital function retrospectively a minimum of 6 months after their surgery. It is possible that patients that did not suffer from any post-operative urological dysfunction might be more prone to better score their post-operative function. Furthermore, despite the scores being based on validated standardised questionnaires, questionnaires themselves are subjective in nature. Ideally, objective measurement tools such as urodynamic studies used in Kim et al's study [27] should be used in conjunction with questionnaires to increase the validity of the results. The results from our study indicate favourable outcomes following robotic TME in relation to FUF. Present evidence of FUF following robotic rectal surgery is extremely limited, with only two studies to date investigating FUF independently to that of males [17, 31]. Of those two, only one, whose sample size is considerably smaller, used a control group (laparoscopy) against its robotic cases and found no difference in outcome between the two groups [17]. Regarding FSF, there was no difference between the two groups in any of the individual components or the composite score. However, due to a large proportion of females being sexually inactive, the sample size of the FSF comparison was very small (nine laparoscopic vs four robotic). This makes any meaningful statistical comparison very difficult. Current evidence on robotic FSF is also extremely limited, being investigated in only three studies [17, 31, 32] of which only one used a control group (laparoscopy). In that study, Morelli et al. [17] found no difference in FSF scores between the two groups. Our study is unique as it has the biggest sample size of its kind. In addition, it is the only study of its kind conducted in the UK, where patient socio-economic background would have no influence on the mode of surgery chosen, since all patients would have been operated in a public healthcare service, the NHS. In contrast to the majority of the previously published literature, the operating surgeons in our study performed fully robotic rather than hybrid procedures [19]. It is possible that the difference in approach could influence the results. This is because damage to the autonomic nerves could occur in two places where dissection is performed laparoscopically during the hybrid procedure. The superior hypogastric plexus can be damaged during dissection around the inferior mesenteric artery pedicle and the hypogastric nerves during mobilisation of the rectosigmoid colon from the gonadals and the ureter [4, 33]. Therefore, in our study, we exploit the full potential of the robotic approach. In addition, all the procedures in both the laparoscopic and robotic cohort were performed by the same two surgeons, thus eliminating the confounding factor of the operating surgeon. Moreover, all the laparoscopic procedures pre-dated the robotic ones, since the operating surgeons shifted their regular practice from laparoscopic to robotic TMEs. It could be argued that any skills acquired during the laparoscopic procedures were transferrable to the robotic ones, implying that any superiority demonstrated for the robotic group was not a result of the surgical approach but due to the advancement of the surgeon's skills. However, this is unlikely considering both surgeons were very experienced laparoscopic rectal surgeons that were trainers for the National Training Programme for Laparoscopic Colorectal Surgery (LAPCO) in the UK. We acknowledge this study is retrospective and non-randomised in nature. However, despite the lack of randomisation, our results demonstrate from the baseline characteristics that the two groups were broadly comparable. The only differences between the two groups (tumour high, neoadjuvant radiotherapy and chemotherapy) are more likely to negatively skew the results against the robotic group, since long-course radiotherapy and lower rectal tumours possess risks for urogenital dysfunction. A limitation of our study was that post-operative urogenital function reporting was taken as a "snapshot", with patients reporting their urogenital outcomes only once in a post-operative period varying from 6 months to 3 years in the laparoscopic group and 8 months to 2 years to in the robotic group. However, unlike other studies, this study does not make any assumptions on the time of recovery of urogenital function but only on the overall post-operative urogenital outcomes. In summary, our study has demonstrated that robotic rectal cancer surgery might offer favourable overall post-operative urological and sexual outcomes in males and urological outcomes in females. This is probably because robotic systems allow for precise dissection across the surgical planes in narrow spaces such as the pelvis, thus enabling preservation of the pelvic autonomic nerves. We acknowledge that there are limitations in the study's design such as being retrospective in nature and open to recall bias. We believe a prospective randomised control trial, focusing on urogenital function, with a bigger sample size that includes urodynamic assessment of urological function is required to establish whether robotic surgery truly offers superior post-operative urogenital outcomes.

TITLE: Effect of Calcium Supplementation on Blood Lead Levels in Pregnancy: A Randomized Placebo-Controlled Trial ABSTRACT.BACKGROUND: Prenatal lead exposure is associated with deficits in fetal growth and neurodevelopment. Calcium supplementation may attenuate fetal exposure by inhibiting mobilization of maternal bone lead and/or intestinal absorption of ingested lead. ABSTRACT.OBJECTIVE: Our goal was to evaluate the effect of 1,200 mg dietary calcium supplementation on maternal blood lead levels during pregnancy. ABSTRACT.METHODS: In a double-blind, randomized, placebo-controlled trial conducted from 2001 through 2003 in Mexico City, we randomly assigned 670 women in their first trimester of pregnancy to ingest calcium (n = 334) or placebo (n = 336). We followed subjects through pregnancy and evaluated the effect of supplementation on maternal blood lead, using an intent-to-treat analysis by a mixed-effects regression model with random intercept, in 557 participants (83%) who completed follow-up. We then conducted as-treated analyses using similar models stratified by treatment compliance. ABSTRACT.RESULTS: Adjusting for baseline lead level, age, trimester of pregnancy, and dietary energy and calcium intake, calcium was associated with an average 11% reduction (0.4 μg/dL) in blood lead level relative to placebo (p = 0.004). This reduction was more evident in the second trimester (−14%, p < 0.001) than in the third (−8%, p = 0.107) and was strongest in women who were most compliant (those who consumed ≥ 75% calcium pills; −24%, p < 0.001), had baseline blood lead > 5 μg/dL (−17%, p < 0.01), or reported use of lead-glazed ceramics and high bone lead (−31%, p < 0.01). ABSTRACT.CONCLUSION: Calcium supplementation was associated with modest reductions in blood lead when administered during pregnancy and may constitute an important secondary prevention effort to reduce circulating maternal lead and, consequently, fetal exposure. BODY: Despite improvements in environmental policies and significant reductions in average U.S. blood lead levels, lead exposure remains a concern for pregnant and lactating women. This is particularly true among certain population subgroups at increased risk, such as women from developing countries and those with occupational exposures [Centers for Disease Control and Prevention (CDC) 2007; Meyer et al. 2003]. In addition, overall declines in environmental sources highlight maternal bone as a long-lived endogenous source of exposure that poses a potential hazard for the developing fetus and breast-feeding infant (Hu and Hernández-Avila 2002). Redistribution of cumulative maternal bone lead stores into the circulation occurs during periods of increased bone resorption, such as pregnancy and lactation (Gulson et al. 2003; Manton et al. 2003; Téllez-Rojo et al. 2004). Prenatal lead exposure has adverse influences on infant birth and neurodevelopmental outcomes across a wide range of exposure (Bellinger 2005; Hu et al. 2006), and maternal bone lead has been shown to be an independent risk factor (Gomaa et al. 2002; Gonzalez-Cossío et al. 1997; Hernández-Avila et al. 2002). The potential role of nutrition in altering susceptibility to lead exposure and toxicity has long been recognized (Aub et al. 1932; Hu et al. 1995; Mahaffey 1974, 1990). Dietary intake concurrent to exposure is known to have an impact on lead dynamics, and nutrients may interact with lead by binding lead in the gut, competing with lead for absorption, altering intestinal cell avidity for lead, and altering affinity of target tissues for lead (Ballew and Bowman 2001). Inadequate calcium consumption has been shown to increase lead absorption (Heard and Chamberlain 1982) and retention (Six and Goyer 1970). Lead competes with calcium at calcium-binding sites and may subsequently alter protein function and calcium homeostasis (Sauk and Somerman 1991). Evidence indicates that low dietary calcium and vitamin D are risk factors for elevated bone lead levels (Cheng et al. 1998). Higher milk intake during pregnancy also has been associated with lower maternal and umbilical cord lead levels in postpartum women (Hernández-Avila et al. 1997), suggesting that calcium status may be an important factor in the maternal–fetal transfer of lead across the placenta. Calcium requirements are increased substantially during pregnancy and lactation in order to meet the needs of the developing fetus and nursing infant for skeletal mineralization and growth (Prentice 2000). Maternal calcium homeostasis is maintained by controlling intestinal calcium absorption, renal calcium excretion, and mobilization of skeletal mineral stores (Kovacs and Kronenberg 1997). The role of dietary calcium and mineral adequacy on skeletal changes of pregnancy and lactation is controversial; however, it is recommended that pregnant and breast-feeding women consume 1,000–1,300 mg calcium per day, depending on their age (Institute of Medicine 1997). In a randomized, double-blind, placebo-controlled trial of 1,200 mg daily calcium supplementation in lactating women, we have previously shown that calcium supplementation reduced maternal blood lead by 15–20% (Hernández-Avila et al. 2003) and breast milk lead by 5–10% (Ettinger et al. 2006) over the course of lactation. Our objective in the present study was to evaluate the effect of 1,200 mg daily calcium supplementation on maternal blood lead levels during pregnancy, the period of greater relevance for maternal–fetal transfer of lead. BODY.MATERIALS AND METHODS.STUDY POPULATION AND DESIGN: We recruited pregnant women from 2001 through 2003 at the Mexican Social Security Institute (Instituto Mexicano del Seguro Social) pre-natal clinics that serve a low- to moderate-income population in Mexico City. We assessed 3,836 women for eligibility, of whom 1,981 did not meet study eligibility criteria (pregnancy of no more than 14 weeks' gestation; not presenting with a high-risk pregnancy; plans to reside in the metropolitan Mexico City area for ~ 5 years) or had other reasons not being enrolled (n = 2). Of the remaining 1,853 eligible women, 670 (36%) agreed to participate and signed the informed consent, and were randomly assigned to receive a daily supplement of 1,200 mg calcium [two 600-mg calcium carbonate tablets (Wyeth Consumer Health Care/Lederle Laboratories, Inc., México City, México) at bedtime; n = 334] or placebo (n = 336). We assessed blood lead levels, dietary calcium intake, and reported use of lead-glazed ceramics (LGC) at three time points: baseline (first trimester), 6 months (second trimester), and 8 months (third trimester). We assessed compliance by pill count at each follow-up visit. We defined women who had at least one blood lead measurement at 6 or 8 months' gestation (n = 565; 84%) as having completed follow-up. Eight women did not have baseline blood lead levels, yielding a total of 557 subjects (83%) available for inclusion in the final analyses (Figure 1). The research protocol was approved by the Human Subjects Committee of the National Institutes of Public Health, the Mexican Social Security Institute, the Brigham and Women's Hospital, and the Harvard School of Public Health and complied with both Mexican and U.S. federal guidelines governing the use of human participants. All participating mothers received a detailed explanation of the study intent and procedures and were advised on identifying and avoiding LGC pottery use during pregnancy before signing the approved written informed consent. BODY.MATERIALS AND METHODS.BLOOD LEAD MEASUREMENT: Blood lead measurements(1.0 μg/dL = 0.0483 μmol/L) were performed using graphite furnace atomic absorption spectrophotometry (Perkin-Elmer model 3000; Norwalk, CT, USA) at the American British Cowdray (ABC) Hospital Trace Metal Laboratory according to a technique described in Miller et al. (1987). The laboratory participates in the CDC blood lead proficiency testing program administered by the Wisconsin State Laboratory of Hygiene (Madison, WI, USA) and maintained acceptable precision and accuracy over the study period. BODY.MATERIALS AND METHODS.BONE LEAD MEASUREMENT: At 1 month postpartum (± 5 days), maternal bone lead was estimated by a spot-source cadmium-109 K-X-ray fluorescence (K-XRF) instrument at the research facility at the ABC Hospital. We used two 30-min in vivo measurements of each subject's midtibial shaft (representing cortical bone) and patella (trabecular bone). The physical principles, technical specifications, validation, and use of the K-XRF technique have been described in detail elsewhere (Chettle et al. 2003; Hu et al. 1998). For quality control, we excluded bone lead measurements with uncertainty estimates > 10 and 15 μg lead/g mineral bone for tibia and patella, respectively. BODY.MATERIALS AND METHODS.DIETARY INTAKE: We assessed maternal dietary intake in each trimester of pregnancy using a semiquantitative food frequency questionnaire designed to estimate usual dietary intake over the prior month. We based the questionnaire on the semiquantitative food frequency questionnaires and validation methodology used in the Harvard Nurses' Health Study and Health Professionals' Follow-up Study (Willett et al. 1985, 1987). We translated the questionnaire and validated it for use specifically for the Mexican Spanish-speaking adult population (Hernández-Avila et al. 1998). BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: We compared baseline characteristics of participants between the calcium and placebo groups using Wilcoxon ranksum (Mann–Whitney U-test) two-sample test of equality or Student's t-test, as appropriate. We performed a similar comparison between those included in the analyses and those lost to follow-up. We evaluated the effect of calcium supplement on blood lead concentration using an intent-to-treat analysis by means of a mixed-effects regression model with a random intercept for each subject. This approach takes into account the within-subject correlation structure attributed to the repeated measurements, yielding valid standard errors of the effect estimates. Blood lead concentrations in the second and third trimester of pregnancy were the outcome variables; however, we used models featuring natural-log– transformed blood lead because this parameterization provided the best fit. In order not to exclude very low blood lead concentrations from the analysis, we substituted 27 blood lead measure ments (1.6% of the total) below the limit of detection (1 μg/dL) with random numbers following a uniform distribution between 0 and 1. We adjusted models for the following baseline variables: first trimester log-transformed blood lead concentration, maternal age (years), treatment group, daily calcium (grams per day) and energy intake (kilocalories per day), and trimester of pregnancy. To assess the overall intent-to-treat effect of calcium supplementation on blood lead concentrations throughout the last two trimesters of pregnancy, we fitted the following model: where ln(BPbi,j) is the loge-transformed blood lead concentration for subject i at trimester j, α + ui denotes the random intercept where ui represents the error term associated to the ith subject [i = (1, 2, . . . , n); ui ~ n(0, σ2u)], Si is a dummy variable that indicates treatment assignment, lnBPbi is the initial (baseline) natural log-transformed blood lead concentration of the ith subject, Tj is the jth trimester of pregnancy [j = (2,3)], Ci is the baseline daily energy intake, Cai is the baseline daily calcium dietary intake, Ai is age, and ɛi,j denotes the random variation [ɛi,j ~ n(0, σ2)]. The overall treatment effect estimate is the coefficient β1. We fitted a second model to estimate the treatment effect at each trimester: where SiTj denotes the interaction term between blood lead levels and trimester of pregnancy. The treatment effect estimate in the second trimester is the coefficient β1, and the effect in the third trimester is (β1 + β7). We used a secondary dose–response study to further assess the effectiveness of supplementation. We assessed compliance by pill count at each visit and analyzed it as proportion of expected pills used between baseline (first trimester) and end of follow-up (8 months' gestation). We defined treatment compliance group in three ways: ≥ 50% of pills consumed, ≥ 67% of pills consumed, and ≥ 75% of pills consumed. To try to disentangle the effect of calcium supplementation on bone lead mobilization versus gastrointestinal absorption, we developed models with an interaction model for postpartum bone lead levels and reported use of LGC. The rationale for fitting this model was that the effect of the supplement may have been larger in those who had larger bone lead concentration and/ or in those who used LGC. We generated a new dummy variable designating high and low patella bone lead levels dichotomized at the median (5.6 μg/g) and created a two-way interaction term with LGC use (yes/no). We did not include a three-way interaction because we found no reason to think that the magnitude of the effect of bone lead concentrations, and thus bone lead mobilization rates, on blood lead would depend on the use of LGC. We fitted the following model: where Pbit is blood lead concentration for the ith subject at the tth trimester, Si is the supplementation group, BPbi is the first available postpartum bone lead measurement, LGCit is current use of LGC in the ith subject at the tth trimester, and δ1 represents the difference in the effect of supplementation between the high and low bone Pb concentration groups, and δ2 represents the difference in the effect of supplementation between the current use/ not use of LGC groups. Covariates are baseline blood lead level, baseline daily calcium dietary intake, baseline daily energy intake, age, and trimester of pregnancy. Because we were trying to disentangle biologic mechanisms, we restricted these models to those who with ≥ 75% compliance. Finally, to account for possible heterogeneity of treatment effects according to initial blood lead levels, we also performed analysis by baseline blood lead group (< 5 μg/dL vs. ≥ 5 μg/dL) using an intent-to-treat analysis and then among only those women with ≥ 50% compliance. We performed all statistical analyses using Stata for Windows (version 9.0; StataCorp LP, College Station, TX, USA). BODY.RESULTS: We randomized 670 eligible women to receive calcium supplementation (n = 334) or placebo (n = 336) (Figure 1). Baseline characteristics were largely similar for both the calcium and placebo groups. Mean maternal age was 1 year higher in the control group (26.9 years) than in the calcium group (25.9 years; p = 0.02) (Table 1). Approximately 35% of women reported current use of traditional LGC for storing, preparing, or serving food; however, we found no significant differences by treatment group. Dietary calcium intake, also not significantly different between the two groups, was about 900 mg/day on average. Geometric mean (and geometric standard deviation) prerandomization blood lead levels were 3.8 (2.0) and 4.1 (2.0) μg/dL for the calcium and placebo groups, respectively (p = 0.05). A total of 565 women (84%) completed follow-up. Comparing the group that completed follow-up (placebo n = 277; calcium n = 288) with those lost to follow-up [placebo n = 59 (18%); calcium n = 46 (14%)], we found no significant differences by treatment group assignment (p = 0.18). Those women who remained in the study reported higher daily energy intake (p < 0.01) and higher use of LGC (p = 0.04) at baseline. Those women who completed follow-up reported higher current use of LGC (36%) than those who did not complete follow-up (26%); among those completing follow-up, however, we found no significant differences in reported LGC use by treatment group. In the intent-to-treat analysis (n = 557), calcium supplementation was associated with an overall average reduction of 11% in maternal blood lead concentrations relative to placebo (p = 0.004) (Table 2). In a secondary analysis, this reduction was more evident in the second trimester (14% reduction, p < 0.001) than in the third trimester (8% reduction, p = 0.107). These results did not change when we controlled for hematocrit level (data not shown). When we assessed the dose–response effect of calcium supplementation for women "as treated" (n = 557) using models stratified by treatment compliance, we saw a clear dose–response effect of calcium on blood lead concentration (Table 3). Among those women who consumed ≥ 50% of pills, calcium was associated on average with a 15% reduction in blood lead levels compared with those taking placebo (p < 0.001). This increased to 19% (p < 0.001) and 24% (p < 0.001) for those who consumed ≥ 67% of pills and ≥ 75% of pills, respectively (p for trend < 0.001). Figure 2 shows the effects of calcium and placebo on maternal blood lead over time among the high-compliance group. Among the low-compliance group (< 50% of pills consumed), blood lead was higher in the calcium-supplemented group, suggesting that these women were somehow different from the low compliers receiving placebo. In fact, in the group that completed follow-up (n = 565), those with low compliance reported higher current use of LGC in the calcium group (35%) compared with placebo (27%), which might explain the apparent increase in blood lead among the low-compliance group. We found no significant differences in reported LGC use among the high-compliance group. Figure 3 shows the proportional reduction [and 95% confidence intervals (CIs)] in blood lead due to calcium supplementation, stratified by use of LGC and patella lead level, among the high-compliance group. Among women consuming ≥ 75% of pills, those with high patella bone lead experienced greater reductions than those women with lower bone lead levels, corresponding to a 23% reduction (p = 0.01) for those with no reported use of LGC and a 31% reduction (p < 0.01) for those who reported use of LGC. In this subset of most compliant women with high patella bone lead (> 5 μg/g) and reported use of LGC, the effect corresponds to an average blood lead reduction of 1.95 μg/dL (95% CI, −0.78 to −2.87). We repeated the analysis by baseline blood lead group (< 5 μg/dL vs. ≥ 5 μg/dL) using intent-to-treat and as-treated analyses among only those women with compliance ≥50% of pills consumed (Table 4). The effects of calcium appeared stronger in the group with higher blood lead at baseline (17% reduction), compared with those with baseline blood lead levels < 5 μg/dL (7% reduction). However, when we restricted the analysis to those women who were more compliant, the reductions were similar between the women with higher (≥ 5 μg/dL = 17%) and lower (< 5 μg/dL = 14%) blood lead at baseline. Among those women with low compliance (< 50% of pills; n = 82), those with low baseline blood lead (< 5 μg/dL) appeared to experience a paradoxical effect of calcium on blood lead levels (an increase of 34%). Those who started the study with higher blood lead (≥ 5 μg/dL) showed the same average effects of treatment (17% reduction), although not statistically significant. Further analysis revealed, however, that the reported use of LGC in low compliers was higher among the calcium group (35%) than in the placebo group (27%), which may account for the apparent differences in treatment effect (7% vs. 17% reduction) observed in the intent-to-treat analysis by baseline blood lead. BODY.DISCUSSION: In this randomized control trial, calcium supplementation (1,200 mg) was associated with modest reductions in blood lead levels when administered during pregnancy. These effects were clearly stronger with increasing compliance, with a 24% average reduction in the most compliant women, and strongest in those with baseline blood lead level > 5 μg/dL (17% average reduction). In the subset of most compliant women with high patella bone lead (> 5 μg/g) and reported use of LGC, we found the greatest reduction in blood lead of 31%, which corresponds to an average reduction of 1.95 μg/dL (95% CI, −0.78 to −2.87). These results are consistent with our previously published randomized trial, which showed that dietary calcium supplementation among postpartum women reduced maternal blood lead by 15–20% over the course of lactation (Hernández-Avila et al. 2003). In that study, the effect among women who were compliant with supplement use (≥ 50% of pills consumed) and had high bone lead (patella lead > 5 μg lead/g bone mineral) was an estimated reduction in mean blood lead of 1.16 μg/dL (95% CI, −2.08 to −0.23). These results are also consistent with the results of a study by Gulson et al. (2004) of blood lead isotopic ratios during pregnancy among women who had recently immigrated to Australia. The authors found that compared with an earlier group of such women they had studied who had calcium-deficient diets, calcium-replete women had a rise in blood lead levels during pregnancy (with an isotopic fingerprint suggesting the lead came from bone) that occurred later in pregnancy and of a smaller magnitude. Although the use of lead isotopic ratios by Gulson et al. (2004) provided very rigorous and precise methodology to their work, the interpretation with respect to calcium supplementation is limited by the small number of women (< 20) in their cases series [and thus limited statistical power to detect an association (Altman and Bland 1995)] and issues of comparability (e.g., the calcium-deficient women were studied at an earlier time and came from Central Europe, whereas the calcium-replete women were studied at a later time, came from Asia, and were otherwise not matched), making the results of our randomized placebo-controlled trial of particular interest. The effect of calcium may be exerted, at least in part, by decreasing bone resorption and the consequent mobilization of maternal bone lead stores. In a case–crossover trial of calcium supplementation during the third trimester of pregnancy, we have previously shown that maternal bone resorption, as reflected by urinary cross-linked N-telopeptide, was reduced by an average of 13.6 nM bone collagen equivalents/mM creatinine (14%) compared with placebo (Janakiraman et al. 2003), indicating that calcium supplementation can suppress maternal bone mobilization. The effects of calcium may also be attributed to decreasing the intestinal absorption of lead and/or increasing the excretion of lead from circulation. In the present study, we did not have prepregnancy bone lead levels, and Mexican laws forbidding potential radiation exposure during pregnancy did not allow us to obtain bone lead measurements during pregnancy. However, our observation in the stratum of women with no reported LGC use—that the calcium effect is greater in those with high bone lead—suggests that, in this population, the effect may have been exerted mainly through inhibiting bone resorption. Average baseline dietary calcium intake for women in our trials of Mexican women was less than the U.S. recommended dietary intake of 1,000–1,300 mg/day for pregnant and lactating women (Institute of Medicine 1997). Levels of dietary calcium intake in our studies were, however, consistent with those reported in the Mexican National Nutrition Survey (Barquera et al. 2003) and in a nationally representative sample of U.S. women of child-bearing age (Lee et al. 2005). Hertz-Picciotto et al. (2000) followed 195 women over the course of pregnancy and found a U-shaped pattern of maternal blood lead concentration across pregnancy. The late pregnancy increases were steeper among women with low dietary calcium intake in both the low and high age groups, suggesting that lead redistribution may be more pronounced among pregnant women in calcium-deficient states. It is possible that high amounts of calcium are needed to counterbalance the nutritional needs of the developing fetus (Johnson 2001). Other genetic, hormonal, or lifestyle factors may also be involved (Ettinger et al. 2007). Nonetheless, dietary calcium intake likely plays a limited, but still important, role in suppressing mobilization of lead from maternal bone and/or decreasing gastrointestinal absorption of ingested lead, thereby decreasing the risk of fetal and infant exposure. Calcium supplementation during pregnancy may also reduce the risk of hypertensive disorders of pregnancy (Hofmeyr et al. 2007) that may also arise secondary to lead exposure (Rothenberg et al. 2002; Sowers et al. 2002) (and thus conferring additional negative effects of lead for both mother and fetus and a potential benefit of calcium supplementation). The risks posed by calcium supplementation at levels approximating recommended daily intakes in this population are negligible. We therefore conclude that dietary supplementation of calcium intake should be considered as a cost-effective means for lowering transgenerational fetal lead exposure. This is particularly important in populations where dietary calcium intake is low. Because bone lead has a half-life of years to decades, women and their infants will continue to be at risk for exposure long after environmental sources of lead have been abated.

TITLE: ABSTRACT.AIMS/HYPOTHESIS: The aim of this study was to determine whether oral dosing with N-acetylcysteine (NAC) increases intraplatelet levels of the antioxidant, glutathione (GSH), and reduces platelet–monocyte conjugation in blood from patients with type 2 diabetes. ABSTRACT.METHODS: In this placebo-controlled randomised crossover study, the effect of oral NAC dosing on platelet–monocyte conjugation and intraplatelet GSH was investigated in patients with type 2 diabetes (eligibility criteria: men or post-menopausal women with well-controlled diabetes (HbA1c < 10%), not on aspirin or statins). Patients (n = 14; age range 43–79 years, HbA1c = 6.9 ± 0.9% [52.3 ± 10.3 mmol/mol]) visited the Highland Clinical Research Facility, Inverness, UK on day 0 and day 7 for each arm of the study. Blood was sampled before and 2 h after oral administration of placebo or NAC (1,200 mg) on day 0 and day 7. Patients received placebo or NAC capsules for once-daily dosing on the intervening days. The order of administration of NAC and placebo was allocated by a central office and all patients and research staff involved in the study were blinded to the allocation until after the study was complete and the data fully analysed. The primary outcome for the study was platelet–monocyte conjugation. ABSTRACT.RESULTS: Oral NAC reduced platelet–monocyte conjugation (from 53.1 ± 4.5% to 42.5 ± 3.9%) at 2 h after administration and the effect was maintained after 7 days of dosing. Intraplatelet GSH was raised in individuals with depleted GSH and there was a negative correlation between baseline intraplatelet GSH and platelet–monocyte conjugation. There were no adverse events. ABSTRACT.CONCLUSIONS/INTERPRETATION: The NAC-induced normalisation of intraplatelet GSH, coupled with a reduction in platelet–monocyte conjugation, suggests that NAC might help to reduce atherothrombotic risk in type 2 diabetes. ABSTRACT.FUNDING:: Chief Scientist Office (CZB/4/622), Scottish Funding Council, Highlands & Islands Enterprise and European Regional Development Fund. ABSTRACT.TRIAL REGISTRATION:: isrctn.org ISRCTN89304265 ABSTRACT.ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00125-012-2685-z) contains peer-reviewed but unedited supplementary material, which is available to authorised users. BODY.INTRODUCTION: Oxidative stress is implicated in both the aetiology and the cardiovascular complications of type 2 diabetes [1–5]. Increased reactive oxygen species (ROS) generation is a feature of platelets in type 2 diabetes [6] and contributes to platelet hyperaggregability associated with the disease [7, 8]. Oxidative stress is also implicit in endothelial dysfunction associated with atherosclerosis and subsequent thrombotic complications [9]. Since the realisation that aspirin fails to show sufficient clinical benefit in primary prevention of cardiovascular events in diabetes to merit its universal prescription to this group of patients [10, 11], there is renewed urgency in finding alternatives. Given the central role of oxidative stress in both diabetes and the resultant cardiovascular complications, antioxidant intervention is a promising therapeutic option that could specifically target atherothrombotic processes. Glutathione (GSH) is an abundant, key endogenous antioxidant that is depressed in platelets from patients with type 2 diabetes, contributing to hyperaggregability [12]. Intramuscular GSH administration increases levels of protective nitric oxide (NO) in platelets from patients with type 2 diabetes, with a concomitant decrease in plasma levels of the fibrinolytic inhibitor, plasminogen activator inhibitor-1 (PAI-1) [13]. However, GSH is a poor candidate for oral therapy because of peptide digestion in the stomach and poor membrane penetration. N-acetylcysteine (NAC), is a well-recognised intravenous therapy used to redress acute GSH depletion in overdosage with paracetamol (known as acetaminophen in the USA and Canada) [14]. NAC is currently under investigation for its antioxidant benefits in a range of clinical conditions [15–20], although there are some doubts about its efficacy, particularly in contrast-induced nephropathy [21]. In the diabetes arena, oral NAC has been shown to improve endothelial function in a rat model of diabetes [22] and to reduce endothelial activation, oxidative stress markers [23] and blood pressure [24] in patients with type 2 diabetes. Very high concentrations (3 mmol/l) augment NO-mediated inhibition of platelet aggregation in blood from obese patients in vitro [25]. Our own previous studies in vitro indicated that much lower concentrations of NAC, which are achievable with oral dosing (10–100 μmol/l), inhibit platelet function in blood from healthy volunteers [26] and in patients with type 2 diabetes [27]. This effect was associated with increased intraplatelet GSH, inhibition of oxidative stress and increased levels of NO metabolites. Here, we tested the hypothesis that oral NAC dosing causes acute (2 h) inhibition of platelet–monocyte conjugation and microparticle count—both recognised as markers of cardiovascular risk—in blood from patients with type 2 diabetes, and that the effect is maintained after daily dosing for 7 days in free-living individuals. Furthermore, we sought to explore the association between the effectiveness of NAC and baseline intraplatelet GSH. BODY.METHODS: Eligible patients with type 2 diabetes (Tables 1 and 2) were recruited through local general practitioners. All patients gave written informed consent in line with International Conference on Harmonisation Good Clinical Practice guidelines. At an initial screening visit, venous blood (9 ml) was collected from the antecubital fossa into lithium heparin-containing tubes (Sarstedt, Leicester, UK) to screen lipid and renal status (Piccolo Lipid and Renal Panels, respectively; Abaxis, Darmstadt, Germany); a finger-prick blood sample was obtained for HbA1c determination (DCA 2000+ Analyser; Bayer, Newbury, UK). Individuals meeting the study inclusion criteria returned within 2 weeks for commencement of the clinical study.Table 1Patient characteristicsCharacteristicAge (years)63.9 ± 10.1Sex (male/female)9:5BMI (kg/m2)33.1 ± 5.7HbA1c (%)6.9 ± 0.90HbA1c (mmol/mol)52.3 ± 10.3Cholesterol (mmol/l)4.7 ± 1.1Data are expressed as mean ± SD (n = 14)Table 2Clinical management regimens for study patientsTreatmentNo. of patientsDiet alone4Metformin alone3Metformin + sulfonylurea3Metformin + sulfonylurea + glitazone3Sulfonylurea alone1 Inclusion criteria for the study were as follows: (1) men or post-menopausal women with type 2 diabetes (to avoid the variability of platelet count and behaviour during the menstrual cycle); (2) those not receiving aspirin (aspirin was withdrawn at least 10 days in advance of the first visit and throughout the study period); (3) those not receiving lipid-lowering therapy—where necessary, statins were withdrawn 1 month in advance of the first visit and throughout the study period. Exclusion criteria for the study were as follows: (1) very poorly controlled diabetes (HbA1c >10% [86 mmol/mol]); (2) significant hypertriacylglycerolaemia (triacylglycerols >4 mmol/l); (3) renal disease, defined by estimated GFR (eGFR) <40 ml/min; (4) current or recently stopped (within last 6 months) smokers; (5) patients receiving other antiplatelet therapy (e.g. clopidogrel, dipyridamole) or lipid-lowering therapy, or where patients were unable or unwilling to stop therapy for the duration of study participation; (6) current use of tetracycline antibiotics or cough suppressants; (7) patients with asthma, because of the low but increased risk of bronchospasm and the increased frequency of anaphylactoid reactions to NAC in asthma suffers. BODY.METHODS.STUDY DESIGN: Patients were randomised to receive either NAC (1,200 mg) or placebo daily for a total of 8 days: both researchers and patients were blinded in this crossover study. On day 0, patients attended the Highland Clinical Research Facility between 09:00 and 09:30 hours, after an overnight fast to minimise diet-induced and diurnal variations in platelet reactivity. A baseline venous blood sample (90 ml) was drawn from the antecubetal fossa using a 21G butterfly device and syringe before oral administration of NAC 1,200 mg (2 × 600 mg capsules) or placebo. The butterfly was flushed with normal saline after initial sampling and 2 h later, having discarded the first 2 ml blood, a second blood sample was taken in the same manner as the first and the patients were provided with a 6-day supply of NAC or placebo capsules for once-daily self-administration. On day 7 of the study, patients returned after an overnight fast for a blood withdrawal before and at 2 h after their final NAC or placebo dose in this arm of the study (protocol identical to day 0). Patients received a light breakfast of buttered toast and water immediately after baseline measures on study visits. A washout period of at least 1 week was allowed before patients returned for the crossover, which followed the same protocol as the first arm of the study. The study design is summarised in Fig. 1 and the recruitment, enrolment and randomisation process is summarised in electronic supplementary material (ESM) Fig. 1.Fig. 1Diagrammatic representation of the study design Clinical Trial Authorisation was granted by Medicines & Healthcare Products Regulatory Agency, Eudract number 2008-001620 and the study was approved by the North of Scotland Research Ethics Committee, study number 06/S0901/39. Clinical trial registration number: ISRCTN89304265. Consistent blood sampling and handling is essential for meaningful results for the proposed flow cytometry assays in particular. Drawn blood was rapidly transferred to tubes containing EDTA, trisodium citrate or heparin (Monovette) and gently inverted several times to mix. Heparinised blood was used for platelet–monocyte conjugation and microparticle experiments, citrated plasma for PAI-1 measures and EDTA for platelet total GSH (tGSH) measurement. Platelet–monocyte conjugates were measured by flow cytometry detection of dual-stained, whole blood samples. Briefly, 100 μl of heparinised whole blood was incubated with CD14-FITC and CD41-PE-Cy5 antibodies, or the appropriate isotype controls, for 15 min. After addition of 1 ml FACS lysing solution (Becton Dickinson, Oxford, UK), the cells were incubated for a further 15 min and then immediately analysed for conjugates using a FACSCalibur flow cytometer (Becton Dickinson). Platelet–monocyte conjugates were identified as CD14/CD41-positive events. Microparticles were prepared from heparinised whole blood. Samples were centrifuged at 500 g for 5 min, the plasma was removed and centrifuged at 5,000 g for 5 min; this was repeated twice to remove any remaining cells. Next, 1 ml of this plasma was further centrifuged at 16,000 g for 5 min, and the microparticle pellet was washed with filtered FACS flow (Becton Dickinson), centrifuged again at 16,000 g and then resuspended in 0.5 ml FACS flow spiked with 3 μm polystyrene latex beads (Sigma Aldrich, Poole, UK). These purified microparticles were acquired through a FACSCalibur flow cytometer and their numbers calculated relative to the known, fixed number of beads acquired at the same time. Plasma was prepared by centrifugation of citrated blood at 1,000 g at room temperature for 10 min and then frozen at −70°C. PAI-1 antigen and PAI-1 activity were both measured using Zymutest ELISA assay kits (Hyphen BioMed, Paris, France) according to the manufacturer's instructions. tGSH was measured in platelet extracts using an established HPLC method. Briefly, platelet suspensions (normalised to 150 × 103/μl) were lysed before disulfide reduction, achieved by the addition of reducing agent (10% tri-butyl phosphine in dimethylformamide; 4°C for 30 min). Samples were deproteinised by the addition of 150 μl trichloroacetic acid (10% in 1 mmol/l EDTA), vortexed (20 s), then centrifuged (8,000 g, 5 min, 4°C). One-hundred microlitres of the supernatant fraction was prepared for derivatisation by the addition of 20 μl of 1.55 mol/l sodium hydroxide, 250 μl of 0.125 mol/l borate buffer (pH 9.5) and 100 μl of 1 mg/ml ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F). Samples were vortexed briefly and incubated (60°C, 1 h). Cooled samples were analysed by HPLC (Agilent 1200 series; Agilent Technologies, Stockport, UK) using fluorescence detection (excitation λ = 385 nm, emission λ = 515 nm). Chromatographic separations were achieved with a Gemini-NX C18 column (250 mm × 4.6 mm × 5 μm; Phenomenex, Macclesfield, UK) and a mobile phase of 7% acetonitrile, 93% 100 mmol/l KH2PO4 at pH 2.1. Forty-microlitre samples were injected onto the column and eluted at a flow rate of 1 ml/min. Thiol derivatives were quantified using peak areas. Protein concentrations in extracts were quantified using a Coomassie protein assay (Perbio Science, Cramlington, UK). Total NAC (i.e. oxidised + reduced) was measured in deproteinised plasma using HPLC with fluorescence detection (as above) according to a method adapted from that of Wilkinson et al [28]. A review of the literature pertaining to the various assays conducted in this project identified platelet–monocyte conjugation as the least powerful technique used in this study. The power calculation was therefore conducted for this measure: for a significance level of p = 0.01 and a power of 80% for detecting a change in platelet–monocyte conjugation of 10%, 15 individuals were recruited to the study. Patient characteristics are expressed as mean ± SD; all other results are expressed as mean ± SEM. Curve fitting and statistical tests were performed using GraphPad Prism software (version 5.00; Graphpad, San Diego, CA, USA). Data distribution was assessed using the Kolmogorov–Smirnov test. All data sets followed Gaussian distribution. It was therefore appropriate to use parametric statistics: t tests, two-way ANOVA with repeated measures and Pearson's correlation tests were used, as appropriate. p < 0.05 was considered to be statistically significant. The investigational product (NAC) and placebo tablets were supplied by Tayside Pharmaceuticals (Dundee, UK). CD14-FITC (BD 555397), IgG2a-FITC (BD 555573), CD41-PE-Cy5 (BD 559768), IgG1-PE-Cy5 (BD 555750) and FACS lysing solution were all obtained from Becton Dickinson. Polystyrene latex beads (3μm) were purchased from Sigma Aldrich (Poole, UK). ELISA kits for the measurement of PAI-1 antigen and activity were from Hyphen BioMed (Paris, France). BODY.RESULTS: The characteristics of the study population are shown in Tables 1 and 2. One patient reported feeling nauseous at times between the two visits on the NAC arm of the study but no other adverse reactions were noted. It was not possible to obtain a blood sample from one of the patients after placebo administration at their first visit so data from this patient was excluded from the majority of the analyses. There was insufficient platelet extract for GSH analysis in samples from two further patients. Platelet–monocyte conjugation was significantly inhibited in patients at 2 h after administration of NAC (conjugated monocytes reduced from 53.1 ± 4.5 to 42.5 ± 3.9% of monocyte population: p = 0.011; Fig. 2a); no significant effect was seen in the equivalent results from the placebo arm of the study (p > 0.05; Fig. 2b). Baseline-subtracted data indicate that depression of platelet–monocyte conjugation was maintained (approximately −10 to –15% compared with placebo) on day 7 of once-daily administration of NAC, both before and after the final administration (Fig. 2c; p = 0.035; two-way ANOVA).Fig. 2Effect of NAC (a; p = 0.011; n = 14 by paired Student's t test) and placebo (b; p > 0.05) on platelet–monocyte conjugation after 2 h and after 7 days of daily oral dosing (c; p = 0.010 for NAC vs placebo; n = 13 by repeated measures two-way ANOVA). NAC, but not placebo, was found to reduce platelet–monocyte conjugation at 2 h after oral dosing and the reduction compared with placebo was significant across the 7 day dosing period (black bars, NAC; white bars, placebo.) Insufficient blood was drawn from one patient at one visit and data from that patient was excluded from (c) In the NAC arm of the study there was a trend towards depression of plasma-borne microparticles that did not reach significance (–31%; p = 0.094; Fig. 3a); microparticles were significantly (+25%; p = 0.048) elevated at 2 h after placebo administration (Fig. 3b).Baseline-subtracted data indicate that this pattern of effect was maintained at day 7 after daily oral dosing; the depression of microparticles in the NAC arm compared with the placebo arm of the study was significant (Fig. 3c; p < 0.016).Fig. 3Effect of NAC (a) and placebo (b) on plasma microparticle count after 2 h. There was a significant increase in microparticle count at 2 h in the placebo arm of the study (p = 0.047 by paired Student's t test) but a trend towards a reduction (p = 0.09) in the NAC arm (n = 14). Over a 7 day dosing period (c), there was a significant inhibition of microparticle count between NAC and placebo (black bars, NAC; white bars, placebo: p = 0.016 for NAC vs placebo; n = 13 by repeated measures two-way ANOVA) Insufficient blood was drawn from one patient at one visit and data from that patient was excluded from (c) Plasma NAC was only detectable in 8/14 patients (1–2 μmol/l) at 2 h after NAC administration. No residual NAC was measured at baseline on the second visit (i.e. ∼24 h after the final self-administered dose; threshold for detection, 1 μmol/l). There was no significant effect of NAC or placebo on plasma PAI-1 antigen or activity at either 2 h after the first dose or after 7 days of oral dosing (p > 0.05). Intraplatelet tGSH was not significantly altered in either the NAC or placebo arms of the study at +2 h (Fig. 4 a,b). However, there was a clear association between the effect of NAC on tGSH after 2 h and the baseline pre-NAC tGSH that was not seen in the placebo arm (Fig. 4c). Subsequent subgrouping of patients into those with baseline platelet tGSH >120 nmol/mg protein ('GSH replete') and those below this cut-off ('GSH deficient'), clearly shows that NAC is only effective in enhancing tGSH in the group that is GSH deficient (Fig. 4d). This effect was not seen in the same subgroupings after placebo (Fig. 4e). There was no association between plasma NAC detection and either change in platelet–monocyte conjugation or intraplatelet tGSH (data not shown).Fig. 4Effect of NAC on intraplatelet GSH. Neither NAC (a) nor placebo (b) had a significant impact on platelet GSH (p > 0.05 for both; n = 12). There was, however, a significant negative correlation between the NAC-induced increase in platelet tGSH at 2 h and the baseline tGSH (c: black symbols; p = 0.0007, r = 0.82, r2 = 0.66; n = 12), which was not reproduced in the placebo arm (white symbols; p = 0.97, r = 0.01, r2 = 0.0002; n = 12). The level at which NAC had no effect was estimated to be 120 nmol GSH/mg protein (c: dotted line; this value was used to subdivide patients into tGSH replete (>120 nmol/mg protein) and deficient <120 nmol/mg protein) for further analysis (d, e). Subgroup analysis for individuals with baseline tGSH of <120 nmol/mg protein (black bar; n = 6) and those above this value (white bar; n = 6) clearly showed that GSH is only increased by NAC administration in those with depleted GSH (d: *p < 0.05; **p < 0.01;***p < 0.001 ); placebo failed to show the same trend (e) Insufficient sample was available from two patients for GSH analysis Platelet–monocyte conjugation was inversely correlated with baseline intraplatelet tGSH (Fig. 5a), but not with HbA1c (Fig. 5b), in participants in this study.Fig. 5Correlation between baseline tGSH (a) and HbA1c (b) and platelet–monocyte conjugation (%). There was a significant negative correlation between tGSH (p = 0.047, r = 0.58, r2 = 0.34; n = 12) and platelet–monocyte conjugation (a) but no correlation between HbA1c and platelet–monocyte conjugation (b: p = 0.225, R = 0.044, r2 = 0.05; n = 14). To convert values for HbA1c in % to mmol/mol, subtract 2.15 and multiply by 10.929. Insufficient sample was available from two patients for GSH analysis BODY.DISCUSSION: This double-blind, randomised, placebo-controlled crossover study provides evidence, for the first time, that oral dosing with NAC effectively inhibits platelet–monocyte conjugation and microparticle count in patients with well-controlled type 2 diabetes. The extent of the effect on intraplatelet GSH was inversely correlated with baseline intraplatelet GSH and, unlike HbA1c, baseline intraplatelet GSH was also inversely correlated with baseline platelet–monocyte conjugation. Platelet–monocyte conjugation is gaining recognition as an effective marker for cardiovascular risk [29–32]. Elevated platelet–monocyte conjugation is indicative of an increased level of circulating platelet activation, which might predispose to thrombus. Moreover, conjugation of activated platelets to monocytes propagates activation of the monocytes themselves [33], increasing the potential for monocyte interaction with the endothelium [34], an early critical event in the atherogenic process [35]. A number of antiplatelet agents [36–38] have been shown to depress platelet–monocyte conjugate formation. Circulating microparticles are cell-derived vesicles that are likewise gaining credence as possible markers of cardiovascular risk because of their elevated levels in a range of cardiovascular conditions [39–43]. While their precise role is still to be fully determined, they appear to be associated with tissue factor and might play a role in thrombosis. On this basis, we selected platelet–monocyte conjugates and microparticles as the principal outcome measures for this study. Both platelet–monocyte conjugate and microparticle measures are depressed by NAC compared with placebo within 2 h of administration and the effect is maintained at day 7 following once-daily dosing on the intervening days. The rapidity of onset of the effect on both markers is perhaps surprising, particularly in light of the fact that NAC is undetectable at the 2 h time point in 6/14 individuals. However, given the known rapidity of both NAC absorption (time to peak (tmax) 0.5–3 h) and incorporation into cellular GSH (tmax ∼1 h), coupled with the wide variability in pharmacokinetics of the drug (t1/2 1.4–3.9 h) [20], both the speed of response and the variability in NAC detection at 2 h are explicable. In the case of platelet–monocyte conjugates, the results indicate that conjugation is a highly dynamic two-way process, the balance of which is disturbed by NAC in favour of disaggregation. With respect to the microparticle data, the results are more complex: at least part of the significant effect of NAC on microparticles is due to prevention or reversal of an increase in microparticle count seen in the placebo arm of the study. The driver for this pro-microparticle effect is unknown, but it might be a simple time-mediated event or could be precipitated by the light breakfast taken by participants between samples. The microparticles measured in this study are not characterised and could therefore be derived from inflammatory or endothelial cells as well as platelets. Further exploratory work is required to identify the driving force behind the change in microparticle effect and how NAC inhibits this process. Plasma PAI-1 was not affected by NAC at any of the time points measured. PAI-1 was measured because a previous clinical study using injected GSH [13] had found that plasma PAI-1 was reduced after treatment. The inference from our finding is that the effect found previously is specific to plasma GSH as opposed to intracellular GSH. Neither plasma NAC nor the increased intraplatelet tGSH, found in at least a cohort of our test group, significantly influenced PAI-1 antigen or activity. The impact of oral NAC on intraplatelet tGSH was not as clear cut as we had found previously in vitro [26, 27]. While the tGSH in the placebo arm was reassuringly consistent, that in the NAC arm showed no change in terms of mean value, but revealed a striking pattern of effect that was dependent on the baseline tGSH: low baseline was associated with a large increase, high baseline with either no change or a small decrease. The association was borne out by correlation analysis from which, despite the relatively small sample size, there was found to be a clear inverse correlation between NAC effect size and baseline tGSH, which was not seen in the placebo arm of the study. We used a linear regression plot to estimate the baseline tGSH level that determines likely 'responders' (tGSH deficient) from 'non-responders' (tGSH replete; >120 nmol/mg protein). Retrospective subgroup analysis of responders and non-responders confirmed that tGSH was only significantly increased by NAC in the depleted group. The consequence of this finding is that NAC is only likely to have a biochemical impact in those patients with depleted tGSH (on the evidence of this study, ∼50% of patients with well-controlled type 2 diabetes). The additional finding that there was an inverse association between tGSH and platelet–monocyte conjugation, suggests that tGSH depletion might be an effective marker of cardiovascular risk. Certainly, the correlation between these factors is much clearer than that between HbA1c and platelet–monocyte conjugation, albeit that the patients in this study had relatively well-controlled type 2 diabetes. Furthermore, were low intraplatelet tGSH to be causal in heightened platelet activation in type 2 diabetes, there is the tantalising possibility that NAC administration is most effective in those individuals who are most at risk of cardiovascular complications. This is a novel finding that, if confirmed in a larger study, could point not only to depleted GSH as a contributory factor in heightened platelet activation in type 2 diabetes, but also to the usefulness of intracellular GSH screening before NAC therapy. A critical role for intracellular GSH in determining the extent of NAC benefit might also explain the lack of consistency in results from studies in a range of clinical conditions [15–21], where intracellular GSH has not been a consideration. In summary, the results of this study indicate that once-daily oral administration of NAC holds promise in primary prevention of cardiovascular complications associated with type 2 diabetes. These features, together with known benefits with respect to hypertension [24] and endothelial function [23], extend the possible therapeutic targets for NAC beyond the confines of type 2 diabetes; patients with metabolic syndrome and other cardiovascular conditions in which oxidative stress and GSH depletion feature might also benefit. The ability of NAC to reduce platelet–monocyte conjugation is linked to the degree of platelet tGSH depletion, a selective property that might effectively target those at highest risk of cardiovascular complications. In the impending era of personalised medicine, patients most likely to benefit from NAC administration could be identified through platelet tGSH measurement in a blood sample taken before treatment. A limitation of this study is that it focused on patients with mainly well-controlled type 2 diabetes (mean HbA1c 6.9 ± 0.9% [52.3 ± 10.3 mmol/mol]). It would therefore be interesting to establish whether similar findings relate to patients with poorer glycaemic control. It is also important to establish whether specific diabetes therapies, such as insulin, impact on platelet–monocyte conjugation, intraplatelet tGSH and the effects of NAC; this would have especially important implications with respect to the relevance of this potential therapy in type 1 diabetes. Finally, the effects have only been determined over a relatively short period (8 days). The impact of chronic therapy requires investigating to confirm that the beneficial effects are maintained throughout a much longer intervention period. The study, though relatively small, was adequately powered for the outcome measures. In conclusion, this study is the first of its kind to show that oral NAC therapy has the potential to reduce cardiovascular risk in those patients with type 2 diabetes who have depleted intraplatelet GSH. There currently exists a paradox in the primary prevention of cardiovascular disease in diabetes: while diabetes is known to increase cardiovascular risk, the principal antiplatelet agent, aspirin, has been found to be ineffective in this patient group and is no longer recommended. This leaves diabetes patients untreated and without an efficacious antiplatelet choice. The current finding is timely in that NAC might represent part of the solution for an unprotected patient population. The results also highlight the importance of individualised therapy for patients with diabetes: NAC may only be effective in that subset of the patient population with type 2 diabetes who are deficient in intracellular GSH. However, the likely effectiveness of the treatment could be screened for in advance through measurement of intraplatelet GSH. In addition, the likely link between oxidative stress, GSH depletion and cardiovascular risk might mean that NAC treatment will target those most vulnerable to cardiovascular disease. An outcome study is now merited to confirm that reduction in platelet–monocyte conjugates is reflected in a reduction in cardiovascular events in this patient group. BODY.ELECTRONIC SUPPLEMENTARY MATERIAL: ESM Fig. 1Diagrammatic representation of the recruitment, enrolment and randomisation process. Patients were invited in batches sequentially until pre-determined study power was achieved and, of the 25 interested patients, the study group was selected on a 'first-come first-served' basis until study power was achieved (PDF 204 kb)

TITLE: A meta-analysis of two randomised trials of early chemotherapy in asymptomatic metastatic colorectal cancer ABSTRACT: This report constitutes a prospectively planned meta-analysis combining two almost identical trials undertaken in Australasia and Canada to study the effect of starting chemotherapy immediately in asymptomatic patients with metastatic colorectal cancer. Patients (n=168) were randomised to receive either immediate or delayed treatment (at onset of predefined symptoms). Australasian patients received either weekly 5-fluorouracil and leucovorin (500 and 20 mg m−2, respectively) (n=59) or the daily × 5 Mayo Clinic schedule (425 and 20 mg m−2, respectively) (n=42). Canadian patients were treated with the Mayo schedule (n=67). Otherwise, the two studies were almost identical in design and each used the European Organisation for the Research and Treatment of Cancer (EORTC) QLQ-C30 instrument for measuring quality of life (QoL). Treatment was continued until 6 months had elapsed or disease progression occurred. Low accrual led to trial suspension before the predetermined sample size for either study was reached. Median survival was not significantly better with immediate treatment (median 13.0 vs 11.0 months; hazard ratio, 1.15; 95% confidence interval (CI) 0.79–1.72; P=0.49). There was no statistically significant difference in progression-free survival (time from randomisation until first evidence of progression after chemotherapy, 10.2 vs 10.8 months; hazard ratio, 1.08; 95% CI 0.71–1.64; P=0.73). There was no difference in overall QoL or its individual domains between the two treatment strategies at baseline or at any subsequent time point. Early treatment of asymptomatic patients with metastatic colorectal cancer did not provide a survival benefit or improved QoL compared to withholding treatment until symptoms occurred. BODY: Colorectal cancer (CRC) is the most common malignancy in Western countries. Primary disease, detected early, is potentially curable surgically. However, about 20% of patients have metastatic disease at initial presentation, and about 50% of all stage A–C disease ultimately recur despite surgery and adjuvant therapy. Most of these patients have incurable metastases. Until recently, the most active cytotoxic agent against metastatic CRC was 5-fluorouracil (5FU) potentiated by leucovorin (LV, folinic acid) (Rustum, 1989). A number of clinical trials and three meta-analyses have shown improved response rates with 5FU–LV compared with 5FU alone (Piedbois et al, 1992; Jonker et al, 2000; Thirion et al, 2004). In addition, quality of life (QoL) can be improved as a consequence of tumour response or stabilisation by chemotherapy (Scheithauer et al, 1993; Glimelius et al, 1995). One of these meta-analyses suggested a greater treatment effect on survival advantage for poor performance status (Eastern Cooperative Oncology Group (ECOG) 2+) patients, with a treatment effect on good performance status patients that was similar (Thirion et al, 2004). In asymptomatic patients, chemotherapy may be given immediately on diagnosis of incurable metastatic disease or may be withheld until symptoms develop. The appropriate timing of the use of 'expectant' chemotherapy has been the subject of debate, especially when the potential toxicity of treatment is taken into account. The Nordic Gastrointestinal Tumor Adjuvant Therapy Group (1992) addressed this issue in a randomised controlled trial in 183 patients with advanced but asymptomatic CRC. The results of this study suggested a survival benefit for patients receiving early treatment with methotrexate and 5FU–LV (MFL), but there were methodological issues: only 57% of patients randomised to expectant treatment received chemotherapy; chemotherapy was not standardised; outcomes were poorer than expected in the expectant treatment group; and no formal QoL analyses were published. In a separate study from Finland, the response rate to an epirubicin, methotrexate and 5-FU combination was much greater in asymptomatic patients with advanced CRC than in symptomatic patients (40 vs 4%, P<0.001), and survival correlated with performance status, but this was a post hoc analysis in a nonrandomised trial (Pyrhonen and Kouri, 1992). Inconclusive results from these two studies and a range of opinions among medical oncologists who treat CRC led us to evaluate whether asymptomatic patients should receive chemotherapy immediately or could defer therapy without compromising outcomes. Two almost identical prospective, randomised controlled trials were undertaken by the Australasian Gastrointestinal Trials Group (AGITG) and the NSW Clinical Oncology Group, and the National Cancer Institute of Canada (NCIC) Clinical Trials Group. The primary aim of both studies was to evaluate the effect of the early use of 5FU–LV on survival of asymptomatic patients with advanced CRC. Quality of life was a secondary outcome. We report here a preplanned, prospective meta-analysis of survival and QoL of the two studies. BODY.PATIENTS AND METHODS.PATIENT POPULATION: Patients were recruited from 17 hospitals in Australasia (Australia and New Zealand) and 17 hospitals in Canada between May 1994 and October 1999. Patients in both studies were required to have histologically proven CRC with local recurrence or metastases not amenable to curative treatment. Patients were required to have no symptoms related to advanced disease, specified as: no pain requiring regular narcotic analgesics; no weight loss over 5 kg (unless related to surgery or other illness); no persistent nausea requiring medication; ECOG performance status of 0 or 1 (Canada: Karnofsky performance status >90%); no obstructive bowel symptoms; no persistent fever related to metastatic cancer; and no other symptom which in the opinion of the clinician was due to progressive metastatic cancer. Other eligibility criteria included: no prior chemotherapy for metastatic disease (patients might have received 5FU-based adjuvant treatment more than 6 months before the development of metastatic disease); evaluable or measurable disease according to the criteria of Miller (Miller et al, 1981); creatinine <150 μm l−1 or creatinine clearance >1 ml s−1; bilirubin <25 μm l−1, aspartate aminotransferase (AST) <2 × upper limit of normal (ULN), alanine transferase (ALT) <3 × ULN; granulocytes >1.5 × 109 l−1, platelets >100 × 109 l−1; no central nervous system metastases; no history of prior invasive malignant disease; and no uncontrolled medical condition that would be aggravated by treatment. Women of childbearing potential had to be not pregnant and agree to adequate contraception for the duration of the study. The study was approved by local institutional ethics committees, and written informed consent was obtained. Pretreatment evaluation included patient history, physical examination, clinical tumour measurement, full blood cell count, electrolytes, urea, creatinine, liver function tests (bilirubin, AST, ALT), albumin, calcium and glucose, chest X-ray, electrocardiogram and radiological investigations as indicated to document evaluable or measurable disease. Serum lactate dehydrogenase measurement was mandated in Australasian patients only. BODY.PATIENTS AND METHODS.RANDOMISATION AND TREATMENT: Patients were stratified according to treatment centre and prior adjuvant chemotherapy (yes or no), then randomised to immediate treatment (to begin within 2 weeks) or treatment delayed until the development of symptoms. The treatment schedule differed in the Australasian and Canadian studies. In Canada, the study allowed only the Mayo clinic schedule: (5FU 425 mg m−2 intravenous (i.v.) bolus on days 1–5, LV 20 mg m−2 i.v. bolus on days 1–5, repeated every 28 days). In Australasia, clinicians chose between the Mayo clinic schedule or weekly 5FU–LV (5FU 500 mg m−2 i.v. bolus and LV 20 mg m−2 i.v. bolus), but nominated the schedule before randomisation (Buroker et al, 1994; Kerr et al, 2000). Every attempt was made to treat all patients for at least 2 months. For patients allocated to delayed treatment, chemotherapy was started when defined criteria were met. These criteria included a decline in performance status to ECOG ⩾2 or Karnofsky <90%, weight loss more than 4.0 kg from the time of study entry, persistent nausea requiring medication, pain requiring regular narcotic analgesics, elevation of bilirubin to >25 μm l−1 due to metastatic disease, elevation of AST or ALT to >3 × ULN, other symptoms due to progressive cancer, development of clinically significant third-space fluid collections or if it was felt that further delays in instituting chemotherapy would be unwise. Dose modifications were undertaken according to the toxicity in the previous cycle. Patients with no toxicity at all could have the 5FU dose increased by 50 mg m−2. Grade ⩽2 nonhaematological toxicity or nadir neutrophil counts ⩾0.5 × 109 l−1 or platelets ⩾50 × 109 l−1 did not require dose modification. Grade 3 nonhaematological toxicity or nadir neutrophil counts <0.5 × 109 or platelets <50 × 109 l−1 warranted 5FU dose reduction by 50 mg m−2. Any grade 4 toxicity warranted reduction by 100 mg m−2. Patients whose measures on laboratory parameters had not returned to pretreatment levels at the time of the next cycle or week of therapy had treatment withheld weekly for a maximum of 2 weeks to allow recovery. BODY.PATIENTS AND METHODS.EVALUATIONS OF PATIENTS: All patients were reviewed monthly throughout the study period with history and physical examination, performance status, full blood cell count and serum biochemistry. Assessments of QoL were completed every 2 months and tumour imaging every 3 months. Patients with responding disease (assessed by standard criteria; Miller et al, 1981) continued on treatment. Treatment was discontinued if there was evidence of disease progression, intolerable toxicity despite dose reductions, completion of six cycles of chemotherapy with best response being only stable disease, or at the patient's request. In both trials, QoL assessment was undertaken every 2 months to provide a direct comparison of QoL with and without chemotherapy. Both studies used the European Organisation for the Research and Treatment of Cancer (EORTC) QLQ-C30, which is a well-validated, comprehensive, core questionnaire consisting of 30 items that assess function, symptoms and overall QoL (Aaronson et al, 1991). The Canadian questionnaire had three more questions than QLQ-C30. Identical questions were combined for the analysis. Additional instruments used in Australasia included the linear analogue self-assessment (LASA) scales of Priestman and Baum (1976), the GLQ-8 scales of Coates et al (1983) and the QL index of Spitzer et al (1981). BODY.PATIENTS AND METHODS.STATISTICAL ANALYSIS: Continuous variables were presented as medians with interquartile ranges. Australasian and Canadian studies were combined in a prospective meta-analysis for the primary outcome of survival and also time to ultimate disease progression (time from randomisation until first evidence of progression after chemotherapy). Risk factors considered in univariate and multivariate analysis of predictors of survival and time to ultimate progression included age, sex, performance status, weight, stage at diagnosis and disease-free interval. Outcomes were analysed with the Cox proportional-hazards regression model (with stratification by trial for combined analysis), with hazard ratio and 95% confidence intervals (CIs) reported. Relevant variables were adjusted for in a multivariate model. Survival curves were presented as Kaplan–Meier plots. All analyses used SPIDA and SAS (versions 7 and 8) software, classified data by intention to treat, and were two-sided with a 5% level of significance. Combined data for baseline-adjusted EORTC QLQ-C30 scores for overall QoL and individual domains of QoL for treatment groups were compared. Quality of life scores are rarely normally distributed, and the data distributions in this study were skewed; presenting means and standard deviations was not appropriate. Therefore, the data were dichotomised into 'good' and 'poor' categories, with proportions and 95% CIs presented graphically for the 'good' QoL scores by treatment group and time. Alternatively, medians and interquartile ranges could have been presented; however, the scales were small and presenting medians would not show subtle differences. The primary objective of each trial was to detect differences in overall survival. The intended combined sample size of 400 patients had 83% power to detect a 15% absolute improvement in 1-year survival from 40 to 55% at a 5% significance level. As both trials collected common QoL data using the QLQ-C30 questionnaire, there was good power (>90%) to detect small differences in QoL from the combined studies. With the final sample size of 168 patients (median follow-up of 57 months), the combined analysis would have an 80% power to detect a 20% improvement in 1-year survival (from 45 to 65%). BODY.RESULTS.PATIENT CHARACTERISTICS AND TREATMENT: Owing to slow accrual, recruitment was suspended before the predetermined sample size for each study was reached. However, the prospective meta-analysis was still conducted as intended. A total of 168 patients were recruited (101 Australasians, 67 Canadians) (Figure 1). Patient demographic characteristics and factors of potential prognostic importance were generally well balanced between the immediate- and delayed-treatment arms (Table 1). The median age was similar across immediate and delayed groups for both studies, although there were more patients aged over 70 years in the immediate-treatment groups. All Canadian patients had ECOG performance status 0, whereas 21% of Australasian patients had ECOG performance status 1 on the basis of comorbidity. All Australasian patients had measurable disease, whereas 26% of Canadian patients had only evaluable, nonmeasurable disease. The most common sites of metastases were liver and lung. Only one patient randomised to the immediate-treatment group did not receive protocol therapy, deciding to defer treatment until the disease had progressed. In the delayed-treatment group, 59 (70%) ultimately received protocol (5FU–LV) therapy. Of the remaining 25 patients in the delayed-treatment group, 12 declined chemotherapy at the decision point and were subsequently managed by supportive care (seven), radiotherapy (one), immediate chemotherapy (one), other chemotherapy in a research study (one), and care unknown (two). Two patients had not developed symptoms by 55 and 57 months after randomisation, respectively. Eight patients died before starting therapy 2.2–53 months after randomisation; of these, seven died of progressive disease and one died of postoperative complications after presenting with acute bowel obstruction related to peritoneal metastases. Three patients were treated with radiation therapy alone. No patients received second-line systemic treatments, since neither irinotecan nor oxaliplatin were available during the study period. All Canadian patients received the Mayo clinic schedule of four-weekly 5FU–LV (Table 2). In the Australasian cohort, 22 of 49 patients in the immediate-treatment group and 12 of 33 treated patients in the delayed-treatment group received weekly 5FU–LV. In the delayed-treatment group, the median time from randomisation to treatment initiation was 6 months (range 1–34) in the Australasian cohort and 4 months (1–28) in the Canadian cohort. Reasons for starting treatment in the delayed-treatment group were onset of symptoms (pain (23%), declining performance status (29%), weight loss (20%), nausea (6%), elevated bilirubin (4%), elevated transaminases (6%)), and significant disease progression without protocol-specified increased symptoms (13%). BODY.RESULTS.TIME TO DISEASE PROGRESSION: The time to disease progression from randomisation to after chemotherapy was similar in the immediate- and delayed-treatment groups for both studies (Table 3). Pooling the results of the two studies by meta-analysis (test for heterogeneity, P=0.047) did not disclose a difference in time to disease progression between the immediate- and delayed-treatment groups. Disease progression occurred at a median of 10.2 months after randomisation in patients receiving immediate treatment, and 10.8 months in the delayed treatment group (hazard ratio, 1.01; 95% CI 0.72–1.41). After adjustment for age, sex, weight, ECOG performance status, prior adjuvant chemotherapy, and baseline QoL, the hazard ratio was similar. BODY.RESULTS.SURVIVAL: In September 2004, the median follow-up time was 55 months, and 163 patients had died (83 in the immediate-treatment group and 80 in the delayed-treatment group). In all, 89% of patients died from tumour progression, 3% from treatment-related toxicity, and 8% from other unrelated conditions. Survival after study entry was similar in the immediate- and delayed-treatment groups for both the Australasian and Canadian studies (Table 3). Pooling the results of the two studies by meta-analysis (test of heterogeneity, P=0.5) did not show a difference in survival between the groups. The 1-year survival was 57% for immediate treatment and 46% for delayed treatment. Median survival was 13.0 months in patients receiving immediate treatment and 11.0 months in the delayed-treatment group (hazard ratio, 1.08; 95% CI 0.79–1.47) (Figure 2). Adjustment for risk factors did not significantly change the hazard ratio (hazard ratio, 1.15; 95% CI 0.77–1.72). Multivariate analysis was conducted with the Cox proportional-hazards model. Age, sex, liver metastases, ECOG performance status, baseline QoL scores, weight, disease-free interval >12 months, stage at first presentation, and prior adjuvant chemotherapy did not statistically significantly influence time to disease progression or survival for immediate vs delayed treatment. BODY.RESULTS.QUALITY OF LIFE: Patients completed QoL assessments every 2 months from randomisation until death. The EORTC QLQ-C30 was common to both the Australasian and Canadian studies. Form completion rates were high at baseline and at each subsequent time point. Quality of life was similar for patients in both treatment arms at baseline. All 168 patients completed questionnaires at baseline, with over 95% of all questions answered. At 12 months, 85% of living, immediately treated patients and 74% of living patients allocated to delayed treatment completed questionnaires. For global health status, a score higher than 75% was considered to indicate 'good' QoL. Figure 3 shows the proportion of 'good' scores over time by treatment group; a proportion of 0.5 indicates that half the patients had QoL scores higher than 75%. Global health status in the immediate treatment group was nonsignificantly lower at all time points, except at 8 months, when QoL was the same. There was no statistically significant difference in overall QoL or any component domain between the two treatment strategies at baseline or at any subsequent time point. The use of the LASA scales of Priestman and Baum (1976), the GLQ-8 scales of Coates et al (1983) and the QL index of Spitzer et al (1981) showed no differences from the results of the EORTC scales. Physical well-being (LASA) was rated high at baseline (median 1 cm on a 10 cm scale) with nonsignificant deterioration over time (median 2.2 cm at 8 months) with no difference between immediate- and delayed-treatment groups (data not shown). Scales for pain, appetite, thought of feeling sick, nausea and vomiting, and overall QoL showed no differences between treatment groups or over time. The proportion of patients with a QL index score of 5 (optimal QoL) fell from 60% at baseline to 40% by 14 months, but with no significant difference between the treatment strategies or over time (data not shown). The immediate- and delayed-treatment groups had no significant difference at any time point in most of the individual domains of QoL in the EORTC and other instruments (ECOG performance status, dyspnoea, loss of appetite, constipation, diarrhoea, pain, fatigue, nausea and vomiting, and physical, role functioning, emotional, and cognitive functioning). The two exceptions were insomnia and social functioning, in which those patients in the immediate-treatment group showed a nonsignificant trend for poorer QoL over time compared with those in the delayed-treatment group. BODY.RESULTS.ADVERSE EFFECTS: Treatment toxicities were as expected for these regimens of 5FU–LV (Table 4), with no statistically significant differences in incidence or severity of worst side effects between immediate- and delayed-treatment groups. Neutropenia occurred in 14% of patients and infection was uncommon (6% of patients overall), with no significant difference between patient groups. Grade 3 or 4 stomatitis occurred in 13% of patients, with no difference between the two arms. Grade 3 or 4 diarrhoea occurred in 20% of patients overall and was more common in immediately treated patients. For Australasian patients, a comparison of toxicities for the two different schedules indicated more grade 3 or 4 diarrhoea with weekly treatment (24 vs 13%), but less grade 3 or 4 neutropenia (3 vs 9%) and stomatitis (2 vs 8%). BODY.DISCUSSION: In this analysis, no survival benefit could be demonstrated from the use of immediate 5FU–LV treatment of asymptomatic patients with metastatic CRC, compared to a policy of watchful waiting and institution of 5FU–LV when symptoms occurred. This result occurred even though 25 (30%) patients allocated to delayed treatment did not receive any chemotherapy. Imbalances in characteristics between groups were minor and had no significant effect. Patients on immediate treatment tended to be older, but adjustment for risk factors including age, performance status, prior adjuvant chemotherapy, and baseline QoL did not change the results of the analysis. Quality of life was not enhanced or adversely affected by immediate treatment. The data show no significant difference in overall QoL or any individual domain of QoL between the two groups, at any time point. Up to the 12-month time-point completion rates of QoL questionnaires were high and almost identical between the two treatment arms, indicating reliability of interpretation. However, beyond 12 months, differences in completion rates between the two study arms make interpretation of QoL data difficult, since reasons for noncompletion may vary between the two treatment strategies. The patient population studied is typical of the cohort of patients with metastatic CRC that present without symptoms. All our patients were of good performance status with 88% of patients overall having ECOG performance status zero. In the delayed-treatment group, the median time to the development of symptoms requiring treatment was 5 months with a very wide range (1–34 months), which was similar to the Nordic trial (179 days, range 5–901 days) (Nordic Gastrointestinal Tumor Adjuvant Therapy Group, 1992). However, the result of our study is at odds with the Nordic study, which showed a longer survival for immediate treatment than for delayed treatment. In that study, patients were allocated to receive immediate treatment with 12 courses of MFL (methotrexate, LV, and 5FU), or to 'primary expectancy', with chemotherapy withheld until symptoms occurred. The survival for immediately treated patients in our study was similar to the Nordic study, but for delayed-treatment patients, outcome data were worse in the Nordic study; patients receiving delayed treatment had shorter symptom-free survival compared to immediate treatment (2 vs 10 months, P<0.001), poorer survival (median 9 vs 14 months, P=0.02), and lower 1-year survival (38 vs 55%, P=0.03). In our study, delayed treatment patients had a median time to start of treatment (similar to symptom-free survival) of 5 months and a median survival of 11.0 months, somewhat longer than the Nordic study. In the Nordic study, two-monthly review may have compromised the outcome for delayed patients since significant health deterioration is more likely to have occurred in the longer review interval compared to our study. Alternatively, subtle differences in characteristics of patients may account for the differences in results of the two studies. At the time our two studies were conceived, we deliberately intended to perform a meta-analysis to improve sensitivity of both QoL and survival assessments. Therefore, each study collected essentially identical data. Unfortunately, it was not considered feasible to include the Nordic study data into this meta-analysis for a number of reasons. Firstly, this was a preplanned analysis, free of any bias as a consequence of knowledge of the individual trial results. Secondly, the Nordic trial was the study that generated the questions being asked in these two trials. Thirdly, no formal comparable QoL assessments were undertaken in the Nordic trial and the chemotherapy used was not standardised in the delayed-treatment arm. However, it is possible to undertake a simplified fixed-effects meta-analysis with the end point of 12-month survival using our two studies as prospective evidence and the three studies as total evidence, since this end point is available for all three studies. The results are presented as Table 5, and show a nonsignificant trend towards improved 12-month survival for immediate treatment (odds ratio 1.63, 95% CI 0.71–3.7, P=0.25). Slow accrual was a problem in our study, preventing it from either definitively supporting or refuting the results of the Nordic study. Many patients and physicians had a preference for either immediate or delayed treatment and this limited accrual to the study, even though there was equipoise among oncologists as to which strategy was appropriate. The small sample size in this study has resulted in insufficient power to detect modest differences in survival and QoL between immediate- and delayed-treatment schedules; by chance, we could have missed a 20% difference in 1-year survival. Nonetheless, the minor trend towards better survival, lack of trend towards better progression-free survival, and lack of effect on QoL with immediate treatment suggests that if real benefits exist for early treatment they are small. This study demonstrates the problems of compliance with a management plan involving delayed therapy in patients with terminal cancers. Only 70% (59 of 84) of patients in the delayed-treatment group actually received the protocol-defined chemotherapy, which was marginally higher than the Nordic study (57%, 51 of 90). One-quarter of our patients not receiving chemotherapy died early, or were too ill to receive treatment, and one-third chose alternative treatments. Such compliance issues would tend to bias this study in favour of immediate treatment. This issue should be considered in planning any future studies of chemotherapy in this context. This study was begun in the early 1990s to address the fundamental question of timing of chemotherapy in patients with asymptomatic cancer. At the time the study was conducted, the thymidylate synthase inhibitors such as 5FU were the only treatments of demonstrated benefit. The introduction of additional agents has expanded the range of options for these patients and debate about the timing of the institution of chemotherapy has been overtaken by a desire to incorporate these new agents to achieve the greatest patient benefit (Simmonds, 2000). However, the newer regimens also bring more toxicity, so the question as to whether asymptomatic patients with advanced CRC should be treated remains unanswered (Simmonds, 2000). Certainly the increased toxicity of irinotecan and oxaliplatin would have greater adverse effects on QoL in asymptomatic patients, which would need to be offset by considerable survival gains to make such treatment worthwhile in this setting. Two recently reported studies throw some light on this issue. The MRC FOCUS randomised study accrued 2135 patients and compared a reference treatment of modified de Gramont schedule 5FU–LV followed by irinotecan upon primary treatment failure, with four other regimens using either second-line combination therapy or first-line combination therapy (Seymour, 2005). None of the combination regimens was superior to the reference treatment in terms of overall survival even though a higher response rate was seen. This study suggests that there is no detriment to use of 5FU–LV as initial treatment in an asymptomatic patient, reserving more toxic combination regimens for second-line management. The LIFE study compared an infusional 5FU–LV regimen alone with the same regimen plus oxaliplatin in 725 patients (Pluzanska et al, 2005). While the combination regimen showed better response rate (54 vs 30%, P<0.0001) and progression-free survival (7.9 vs 5.9 months, P<0.0001), there was no difference in survival (15.9 vs 15.2 months). Again, these data suggest that 5FU alone is acceptable as initial management in advanced colorectal cancer, particularly since QoL is likely to be more greatly affected by a combination regimen. To date, data on the effect of palliative chemotherapy on QoL in CRC are insufficient (Conroy et al, 2003). A large, randomised controlled trial in asymptomatic patients would be needed to establish the benefit of newer treatments on patient and cancer outcomes in this subgroup. In the meantime, the results of the study reported here suggest that immediate treatment of asymptomatic patients with 5FU–LV is not necessary to maximise QoL or survival, and that close monitoring and chemotherapy when symptoms prevail is a reasonable management strategy.

TITLE: Placebo can enhance creativity ABSTRACT.BACKGROUND: The placebo effect is usually studied in clinical settings for decreasing negative symptoms such as pain, depression and anxiety. There is interest in exploring the placebo effect also outside the clinic, for enhancing positive aspects of performance or cognition. Several studies indicate that placebo can enhance cognitive abilities including memory, implicit learning and general knowledge. Here, we ask whether placebo can enhance creativity, an important aspect of human cognition. ABSTRACT.METHODS: Subjects were randomly assigned to a control group who smelled and rated an odorant (n = 45), and a placebo group who were treated identically but were also told that the odorant increases creativity and reduces inhibitions (n = 45). Subjects completed a recently developed automated test for creativity, the creative foraging game (CFG), and a randomly chosen subset (n = 57) also completed two manual standardized creativity tests, the alternate uses test (AUT) and the Torrance test (TTCT). In all three tests, participants were asked to create as many original solutions and were scored for originality, flexibility and fluency. ABSTRACT.RESULTS: The placebo group showed higher originality than the control group both in the CFG (p<0.04, effect size = 0.5) and in the AUT (p<0.05, effect size = 0.4), but not in the Torrance test. The placebo group also found more shapes outside of the standard categories found by a set of 100 CFG players in a previous study, a feature termed out-of-the-boxness (p<0.01, effect size = 0.6). ABSTRACT.CONCLUSIONS: The findings indicate that placebo can enhance the originality aspect of creativity. This strengthens the view that placebo can be used not only to reduce negative clinical symptoms, but also to enhance positive aspects of cognition. Furthermore, we find that the impact of placebo on creativity can be tested by CFG, which can quantify multiple aspects of creative search without need for manual coding. This approach opens the way to explore the behavioral and neural mechanisms by which placebo might amplify creativity. BODY.INTRODUCTION: The potency and mechanism of the placebo effect are increasingly researched. The placebo effect is due to the psychobiological phenomena that occur with the administration of the inert substance[1]. These psychobiological phenomena can be induced by expectation, verbal suggestions and classical conditioning [2–4] [5, 6]. Most studies of placebo so far have been in clinical settings with the goal of decreasing negative symptoms such as pain, depression and anxiety. These studies suggest several neurobiological pathways for placebo, which can be differentially activated in different contexts[7]. Analgesia placebo, the best understood placebo to date, is characterized by activation of endogenous opioids and dopamine to reduce spinal nociceptive responses[8]. This pathway provides evidence of how high-order processes—such as expectation—can regulate immediate peripheral sensations such as pain[9]. Placebo has also been widely studied in the treatment of Parkinson's disease. Here, placebo involves the dorsal striatum, which plays a role in motor control, and dopamine release in the ventral striatum, which is part of the reward system[10]. Activation of the reward system has also been shown to affect immune states in mice[11]. There have been much fewer studies on using placebo outside of the clinic in order to enhance positive aspects of performance or cognition. Several studies showed that placebo can improve sports performance (reviewed in [9, 12]). Most of these studies were on professional athletes and used an inert substance or treatment together with suggestions. The athletes were told they were receiving an ergogenic aid (anabolic steroids, caffeine etc.), when in fact they received a placebo. They were then tested for their endurance or strength in the relevant field. Some studies administered an active substance alongside the placebo and some administered only placebo but manipulated expectations regarding its effect. In diverse athletic fields ranging from anaerobic sprint runs and weightlifting to long-range aerobic endurance cycling, placebo extended performance in an expectation-dependent manner. For example, if subjects expected a higher dose of caffeine, they had higher performances, and if they expected negative effects of the substance, performance worsened [13–16]. Pre-conditioning strengthened the placebo effect. For example, subjects received a placebo said to be caffeine, and then were tested for lifting a weight which was reduced without their knowledge. Then they received the same placebo-caffeine, and this time tested on the original weight. Performance was improved relative to a group which received placebo-caffeine in one session only [17, 18]. Several other studies tested the ability of placebo to enhance cognitive performance. In these studies there was no comparison to an active substance. Instead, the independent variable was subjects' expectations manipulated by means of suggestion: a group told that a sham drug or intervention will improve performance (placebo group) is compared to a group going through the same procedure with no mention of improvement (control group). For example, Parker and colleagues showed that an inert substance presented as a drug that acts as a cognitive enhancer increased performance on a prospective memory task, compared with a group which received the same substance and was told it was an inactive control. Prospective memory improvement was at the expense of response times in an ongoing task performed in parallel, indicating increased cognitive effort[19]. Oken and colleagues showed that an inert pill, which was said to be a cognitive enhancer, improved various cognitive abilities in healthy seniors. The control group were same-age subjects which went through the same procedures but were not given the pills. Regression analysis indicated that expectancy, self-efficacy and perceived stress were significant predictors for placebo-related improvement[20]. Placebo was also found to enhance performance in subconscious cognitive tasks. For example, performance on the Stroop effect, a classical response-time test in cognitive psychology, was improved by a sham EEG[21] or by verbal suggestion[22]. More specifically, in the former study, de Gama et al used a within-subjects design that compared performance on the Stroop task at baseline versus -performance during sham EEG said to modulate participants' visual ability to perceive colors: either to enhance it or to decrease it. Participants exhibited less or more interference from written color words in accordance with the corresponding suggestion[21]. In another subconscious implicit learning trial, Colagiuri et al told subjects that an odor influences performance, either positively (placebo group 1), negatively (placebo group 2), or not at all (control). Subjects completed the task in alternating blocks in which odor was presented or not presented. The study found that reaction times on cued trials were faster or slower according to the placebo suggestion[23]. Finally, Weger et al. used a sham subliminal priming procedure which was said to unconsciously enhance subjects' knowledge. Performance on a general knowledge test was enhanced compared to a control group [24]. Based on these encouraging findings, there is scope to explore placebo for improving additional positive aspects of human performance. Here, we ask whether placebo can enhance creativity. Creativity is the ability to generate ideas, solutions or insights that are new and potentially useful[25–27]. Creativity is often viewed as a trait characteristic of a person; however, creativity can also be viewed as a state, affected by expectation and motivation[28–30]. Motivation appears to be a central factor in creative performance[25, 31–33], a finding which is hopeful because motivation can be bolstered, for example, by enhancing belief in one's own competence[34, 35]. In this regard, Green et al found that a suggestion to be more creative increased novelty scores in a word association task, and several studies indicated that conditions that reduce inhibitions can enhance performance on creativity tests[28, 36–39]. There is much current interest in finding ways to enhance the creativity of individuals and groups [40–42]. One obstacle in the study of creativity is the lack of experimental paradigms that allow automated and multi-dimensional views of the creative process. Current paradigms, such as the alternate uses test (AUT) and the Torrance test of creative thinking (TTCT), require laborious manual coding, and do not allow access to the process or intermediate steps by which solutions are reached. A recent advance presented an automated test for creativity called the creative foraging game (CFG, [43]). The CFG is a computer game in which participants search for interesting and beautiful shapes in a well-defined geometric space. This test allows measurement of multiple aspects of the search including fluency, uniqueness of solutions and the length and timing of the paths taken to reach the solutions. Here, we hypothesize that a placebo that combines an inert substance with a verbal suggestion aimed at increasing creativity and reducing inhibitions will increase subjects' creativity. We used an odorant as an inert placebo substance, which is less invasive than pills or injections and hence appropriate for non-clinical settings. We tested creativity with two standard manual tests, AUT and TTCT, and with the automated creative foraging game to compare aspects of the creative search between groups that smelled the odorant with and without the suggestion. BODY.METHODS.PARTICIPANTS: Participants were recruited between March 2014 and June 2015 via social media groups dedicated to recruiting subjects for experiments, and were mostly students from nearby universities. Compensation was 40 NIS (about 10$) per hour. Ninety-six participants took part in the experiment. Before analysis, we removed three participants who were subject to another suggestive experiment directly before the current one, two participants with abnormally short games and one participant who had previous knowledge of the research hypothesis. Data from ninety participants entered the analysis. All participants were blindly assigned to placebo (N = 45) or control (N = 45) groups prior to their arrival in the laboratory. There were no significant age and sex differences between the two groups (placebo: 49% males, mean age 25.4±2.5, age range 20–33; control: 49% males, mean age 26.1±3.2, age range 21–37). Of these 90 participants, 57 participants (30 from the control group and 27 from the placebo group) completed two additional creativity tests (see study design). Here too, no age and sex differences were found (placebo: 52% males, mean age 25.8±2.6, age range 22–33; control: 50% males, mean age 26.2±3.1, age range 21–32). This study was approved by our Institutional Review Board, Wolfson Medical Center Helsinki Committee. All patients provided written informed consent. BODY.METHODS.STUDY DESIGN: The experiment took place in an olfactory research lab, in which experiments typically ask subjects to rate odors and perform tasks. Participants signed informed consent, came into the experiment room, were presented with an odorant in a jar and were asked to smell it, and to rate its pleasantness, familiarity and intensity. Importantly, the placebo group was also told that the odorant is "a unique odor, developed in our lab, which increases creativity and lowers inhibitions". All aspects of the experiment, except the suggestion, were identical between the two groups; the only thing that differed between their experiences was what we told them about the nature of the substance. Following the odorant presentation, the participants were introduced to the creative foraging game (CFG), and played it for 10 minutes. Fifty-seven participants (30 from the control group and 27 from the placebo group) continued the experiment as followed: They were asked to smell and rate the odorant again, the placebo group were told this was to maintain the effect of the odor and the control group were told it was to get odor ratings at different time points, and then completed two classical creativity tests—the alternate uses test (AUT) and a subset of Torrance test of creative thinking (TTCT), in a counter-balanced order, each for 10 minutes as well. The total duration of the experiment was approximately 40 minutes. BODY.METHODS.THE ODORANT: We used a component of the odor of cinnamon (cinnamaldehyde, Sigma-Aldrich, CAS 104-55-2). Participants rated the odor on a visual analog scale as mildly pleasant (normalized scores to a scale of 1–100: 64.2 ± 22.8), mildly familiar (65.5 ± 25) and relatively intense (73.3 ± 16.4). There were no significant differences between the placebo and control groups in odor ratings (pleasantness: MW U = 950, p = 0.61; two-tailed t88 = 0.54, p = 0.59; familiarity: MW U = 989, p = 0.85; two-tailed t88 = 0.15, p = 0.89; intensity: MW U = 776, p = 0.06; two-tailed t88 = 1.85, p = 0.07, a trend towards the placebo group to perceive the odor as more intense). This result rules out the possibility that differences in group performance were due to differential perception of the odor. There was no significant correlation between odor ratings and performance on the creativity tests (S1 Table). There is evidence that cinnamon odor increases attention and memory[44, 45], although conflicting evidence[46] was also reported. Since both groups were equally exposed to the odor, any presumed effect of the odor itself would affect both groups. BODY.METHODS.CREATIVITY TASKS: We used three creativity tests: the Creative Foraging Game (CGF[43]) in which participants are asked to search for interesting and beautiful shapes in a defined geometric space, the Alternate Uses Test (AUT[47]) which is a verbal test for divergent thinking, and the figural Torrance test (TTCT[48]) which is a visual test for divergent thinking. Scoring of all tests was blind for condition. BODY.METHODS.CREATIVE FORAGING GAME (CFG): The creative foraging game (CFG,[43]) is a computer game in which participants create shapes by moving one of ten identical squares at each step, keeping the squares connected by an edge (see Fig 1a). The initial condition is ten squares in a horizontal line. Participants are asked to explore the space of possible shapes (a total of 36,446 shapes), and when they come across a shape they find interesting or creative, to select it to a gallery. This is done by pressing a gray square at the top-right side of the screen, which saves the current shape to the gallery (Fig 1a). The gallery has no limit on the number of shapes. After the game, players perform another task—choosing the five most creative shapes from their gallery. This task is not analyzed in the current study. The automated algorithm allows constant recording and analysis of players' moves and reaction times. 10.1371/journal.pone.0182466.g001Fig 1Creative foraging game (CFG).a: CFG interface. Left: starting point; right: example of a shape. b: Exploration phases are followed by exploitation phases in which participants usually find shapes with shared perceptual meaning. Purple: numbers; green: airplanes. Data for fig 1b is from one of the participants from Ref [43]. We use the method described in Hart et al [43] to analyze the CFG. During the game participants alternate between two phases termed exploration and exploitation[43]. The start and end time points of these phases are automatically defined by a segmentation algorithm. The algorithm uses the time points of choosing gallery shapes as the input. Briefly, exploration phases are defined by increasing time intervals between gallery choices and exploitation phases are defined by decreasing time intervals between gallery choices (see ref [43] for details). Exploration phases can include a single gallery shape. As shown in Ref [43], gallery shapes found in a given exploitation phase have shared perceptual meaning, and are consistently re-discovered by different participants. By clustering the shapes that are re-discovered by different participants, Hart et al. defined shape categories of meaning (SCM). Three examples of SCMs are shapes that resemble airplanes, numerical digits and letters (Fig 1b). Other SCMs include more abstract shapes with visual similarity. In Ref [43], 14 shape categories of meaning were found and were reliably distinguished in a separate discrimination experiment in which people who did not play the game classified shapes from the same SCM versus shapes from another SCM. CFG Scoring: In creativity tests such as AUT and TTCT, experimenters have access to the solutions provided by the participants verbally or in drawn form, but not to intermediate steps or solutions. We reasoned that such forwarded solutions correspond, in the CFG, to the gallery shapes found in exploitation phases, and thus we scored exploitation gallery shapes rather than all gallery shapes. Fluency was scored as the total number of gallery shapes found by a player in all exploitation phases. Originality was scored as follows: each shape in the CFG was assigned a probability to be found, based on a database of 100 games by participants who did not take part in the current study[43]. The originality score of a participant is equal to the average of the minus log of the probabilities of all gallery shapes found in that player's exploitation phases. The flexibility score of a player is the number of different SCMs found by that player based on the SCMs found in a combined dataset of the 100 players of Ref [43] plus the 45 players in the group tested (control or placebo), plus the number of exploitation gallery shapes that did not fall into any of the SCMs. The out-of-the-boxness (OB) score for each participant was the fraction of exploitation gallery shapes that lie outside of the standard set of SCMs defined by the 100 players of Ref [43]. To test the robustness of this result, we used a random subset of 75 out of the 100 players of Ref [43] as a database for the SCM, and found similar results regarding higher OB in the placebo group. It was not possible to use less than 75 players because it is no longer possible to detect SCMs. Note that OB differs from originality because OB considers categories of shapes rather than specific shapes. OB differs from flexibility because it concerns the fraction of gallery shapes outside the SCMs, rather than the number of different SCMs found. BODY.METHODS.CREATIVE FORAGING GAME (CFG).ALTERNATE USES TEST (AUT): We followed the protocol of Ref [49]. Participants were given a list of five common items (shoe, pin, sheet, nail and button) and asked to list as many alternate uses as possible for each object within 10 minutes, while trying to think of original uses (the most common everyday use was indicated in parenthesis). Only responses that did not reiterate the given common uses were counted and included. Suggested uses which were meaningless were discarded. Scoring included fluency, originality and flexibility. AUT scoring: We followed the procedure of Ref [49]. Fluency was scored by the number of alternate uses found for each object averaged over the five objects. Originality was defined as statistically infrequent responses of all responses provided per object. Specifically, for each object, a list of all obtained uses was collected from all participants. Two raters grouped similar uses into groups, with inter-rated agreement of 89.5% (Kappa coefficient 0.79). For example button as an earring/ as jewelry were grouped together, as were shoe to throw at someone/ as a weapon/ to hit someone. An infrequency score was assigned to each group as follows: answers which were listed by 5% or more of the participants were given a score of 0; answers provided by 2–5% were scored 1, and answers less frequent than 2% were scored 2. The total originality score of a participant is the mean over the infrequency score of all responses. Flexibility was scored by the number of different categories (groups) of the solutions of each object. The total flexibility score is the mean over the number of categories for each object of that participant. BODY.METHODS.CREATIVE FORAGING GAME (CFG).TORRANCE TEST OF CREATIVE THINKING (TTCT), FIGURAL PART, CIRCLES SUBSET: We followed the protocol of the TTCT manual[50]. Participants were given a printed page with 35 identical circles with 1.27 cm (0.5 inch) radii in a 5x7 array. They were asked to draw as many different drawings/ideas as possible within 10 minutes, while trying to make each drawing/idea original and creative. Each drawing must include at least one circle and must be given a title. As before, scoring included originality, flexibility and fluency. Fluency was scored by the number of drawings generated from the circles. Originality and flexibility were both scored based on the TTCT scoring guide[50], which specifies originality scores (0, 1, 2 or 3) for about 150 potential drawings, in about 60 different categories. According to the guidelines, a drawing which is not specified in the manual gets 3 points for originality. As before, the total originality score of a participant is the mean over the originality score of all his/her drawings. When two or more circles are combined to create a single drawing, they are given a bonus originality score, which is added, according to the guidelines, to the total originality score. Flexibility was scored by the number of different categories of the drawings of each participant, using the manual. A drawing which was not mentioned in the manual was given a category according to those specified in the manual, or provided with a novel one, if none would fit. The total flexibility score is the mean over the number of categories of that participant. BODY.METHODS.STATISTICAL ANALYSIS: To compare the control and placebo groups, we used the Mann-Whitney (MW) non-parametric test, which is appropriate because the data is not necessarily normally distributed. We note here also results for the historically more widely used two-tailed t-test: In all cases where we compared groups of total size N = 90, the t-test gave results almost identical to the MW test: when MW was significant p<0.05 so was the t-test, and when MW>0.05 so was the t-test. In cases where N = 57 (comparison with the AUT and TTCT tests), T-test results sometimes showed a trend p = ~0.1 when the MW test showed significance or trend for significance p<0.06. These cases are AUT originality: MW p = 0.048, T-test p = 0.11; AUT fluency MW p = 0.056, T-test p = 0.12; AUT flexibility MW p = 0.06, T-test p = 0.11. All statistically significant results reported here employed the Benjamini-Hochberg multiple-hypothesis correction using FDR = 0.15. BODY.RESULTS: The comparison between the placebo and control group is shown in Table 1. We find no significant effect of placebo on the TTCT test, and hereafter we focus on the CFG and AUT tests. 10.1371/journal.pone.0182466.t001 Table 1 Performance of the placebo and control groups in the three creativity tests. Test Measure Placebo median ±MAD Control median ±MAD p-value MW Effect size Creative Foraging Game (CFG) Originality 7.0±0.5 6.7±0.4 0.036 * 0.46 Fluency 14±6.6 16±8.6 0.73 -- Flexibility 10±7.2 11±6.5 0.96 -- Out-of-the-boxness 0.47±0.17 0.35±0.15 0.008 ** 0.6 Exploitation phases 5±2 5±2.2 0.7 -- Alternate Uses Test (AUT) Originality 3.6±1.3 2.6±1.5 0.048 * 0.43 Fluency 4.2±0.9 3.2±1.1 0.056 † 0.43 Flexibility 3.8±0.8 3±1.1 0.06 † 0.44 Torrance Test of Creative Thinking (TTCT) Originality 28±13 28±9 0.75 -- Fluency 10±5 8.5±4 0.34 -- Flexibility 7±3 7±3 0.78 -- ** p value < 0.01; * p value < 0.05; † trend, p value < = 0.06 BODY.RESULTS.THE PLACEBO GROUP SHOWED HIGHER ORIGINALITY: We first consider originality—the extent to which a player finds solutions not found by other participants (Methods). Participants in the placebo group showed significantly higher originality than the control group, both in the CFG (MW U = 753, p = 0.036; Fig 2, left panel), and in the AUT (MW U = 278, p = 0.048, Fig 2, right panel). Effect size was medium (CFG: Cohen's D = 0.46; AUT: Cohen's D = 0.43). 10.1371/journal.pone.0182466.g002Fig 2The placebo group showed significantly higher originality in both the CFG (left) and AUT (right).Scatterplots of originality scores of the placebo (orange) and control (blue) groups. Y axis is originality scores, ranked from lowest to highest. X axis is group ranking (in the AUT since number of participants in the placebo group was smaller by 3, matching is from the highest score onwards). Insets show mean originality in each group, error bars are standard error of the mean. * p value < 0.05. BODY.RESULTS.FLUENCY AND FLEXIBILITY DID NOT SIGNIFICANTLY DIFFER BETWEEN THE GROUPS IN THE CFG, AND WERE MARGINALLY SIGNIFICANT IN THE AUT, IN FAVOR OF THE PLACEBO GROUP: We next compared fluency, defined as the overall number of solutions. In the CFG, we defined fluency as the number of shapes selected to the gallery during exploitation phases, finding no significant difference between control and placebo (MW U = 970, p = 0.73). In the AUT test, fluency showed a trend for being higher in the placebo group than in the control group (MW U = 286, p = 0.056, Cohen's D = 0.43) (Fig 3). 10.1371/journal.pone.0182466.g003Fig 3The placebo group showed a trend for higher fluency in the AUT (right) and no such difference in the CFG (left).Scatterplots of fluency scores of the placebo (orange) and control (blue) groups. Y axis is fluency scores, ranked from lowest to highest. X axis is group ranking (in the AUT since number of participants in the placebo group was smaller by 3, matching is from the highest score onwards). Insets show mean fluency in each group, error bars are standard error of the mean. † p value < 0.06. We also compared flexibility between the placebo and control groups, defined as the number of categories of suggested solutions. We find that flexibility in the CFG was not statistically different between the placebo and control groups (MW U = 1005, p = 0.96). In the AUT, the placebo group showed a trend towards higher flexibility (MW U = 287, p = 0.06). BODY.RESULTS.THE PLACEBO GROUP SHOWED GREATER OUT-OF-THE-BOXNESS IN THE CFG: When comparing flexibility of the two groups in the CFG, we noticed that the placebo group found many shapes that did not fit any category previously discovered by a database of 100 games[43]. We devised a score for this effect, the extent to which players found categories of shapes that were non-standard, naming it "out-of-the-boxness", OB. We asked, for each participant in our study, what fraction of the gallery shapes lies outside of the standard set of shape categories (SCM, see Methods). Note that OB differs from originality because it considers categories of shapes rather than specific shapes. We find that the OB of the placebo group was significantly higher than the control with medium-to-large effect size (MW U = 683, p = 0.008; D = 0.6). Fig 4 shows that this effect seems to be spread among all 90 players. 10.1371/journal.pone.0182466.g004Fig 4The placebo group displayed higher "out-of-the-boxness" (OB) than the control group in the CFG.Main plot is a scatterplot of OB scores of the placebo (orange) and control (blue) groups. Y axis is the fraction of shapes outside standard categories, ranked from lowest to highest. X axis is group ranking. Inset shows mean OB in each group, error bars are standard error of the mean. ** p value < 0.01. BODY.DISCUSSION: We find that placebo can enhance the originality aspect of creativity. We used an odorant together with a verbal suggestion that it enhances creativity and reduces inhibitions. We evaluated creativity using three tests, the classical AUT and TTCT tests and the CFG, an automated creativity test in a well-defined search space. The placebo group showed increased originality in the AUT and CFG, but not the TTCT, by finding solutions rarely found by other players. The placebo group also showed higher out-of-the-boxness in the CFG by finding solutions not included in categories found by a database of 100 players from a previous study. The size of the significant effects was medium to strong, and effects were distributed among most members of the placebo group. Placebo did not seem to strongly affect fluency, the number of solutions found, which suggests that subjects were not simply less selective, but rather genuinely more original. What are the psychological mechanisms that allow placebo to increase the originality aspect of creativity? There are at least two possibilities. The first mechanism is based on extensive research by Amabile and Deci and Ryan[25, 33, 34, 51–53], that suggests that creativity is modulated by motivation. Extrinsic motivators were shown to be mostly detrimental to creativity, whereas intrinsic motivation is conductive to and strongly associated with creative abilities[25, 32, 33, 51, 54–56]. A key factor in intrinsic motivation, according to self-determination theory [34, 35], is the belief in one's competence. For example subjects who practiced encouraging statements (related to self-confidence, releasing anxieties etc.) and omitted self-incapacitating statements showed improved creativity scores[57]. This is in line with the verbal suggestion in our study that the odorant increases creativity, which may have made subjects feel more competent. Additional components of intrinsic motivation, such as social relatedness, may also have been increased by experimenter effects in the present study, by the experimenter's perceived interest in the effects of the odorant. A second possible psychological mechanism of placebo, as suggested by Weger et al., is to weaken inhibitory mechanisms that normally impair performance[24]. Creativity was found to increase in several studies that tested conditions with reduced inhibitions, such as alcohol consumption[36–38]. Wieth and Zacks showed that creative problem solving was improved when participants were tested during non-optimal times of day, and suggested that this is due to reduced inhibitory control[39]. Moreover, studies which used non-invasive brain stimulation by means of transcranial direct current stimulation (tDCS) found enhanced creativity, and attributed it to reduced inhibitions and diminished cognitive control[49, 58]. This effect was suggested to be in line with paradoxical functional facilitation theory, which attributes improved performance of damaged nervous system to release from inhibition[59]. Informal notions in improvisation theatre suggest that the inner critic is a source of inhibition that limits creativity[60]. The verbal suggestion made in our study that the odorant increases creativity and reduces inhibitions may thus work through a reduced-inhibition mechanism and/or by increasing belief in one's competence. Future work can test which of these mechanisms is at play. Our use of three tests for creativity allows a comparison between the different aspects of creativity within and across tests (S2 table). The two manual tests, AUT and TCTT, did not show significant correlation with each other across participants in fluency, flexibility or originality (all spearman coefficients R < 0.17, all p > 0.22). A similar lack of correlation between different manual tests for divergent thinking was reported in previous studies[61–65]. This suggests that each test might measure different aspects of creativity. Encouragingly, we find that the AUT and CFG showed similar results in terms of increased originality by the placebo group, with a smaller effect on fluency. Whereas fluency and originality did not show significant correlation across tests, when correlating the tests using a combined Z-score for fluency and originality, a moderate and significant correlation is found between CFG and AUT (R = 0.27, p = 0.04,[43]). This suggests that the CFG may complement the AUT as a useful test for creativity, with the advantage that CFG is an automated analysis that does not require manual scoring. It is interesting to note in this regard that in terms of the creative product, the CFG and TTCT are both figural tests, whereas the AUT is verbal. Yet, here the placebo effect was beneficial for the CFG and AUT, but not TTCT. Whereas the reason to this difference is not clear to us, enhanced creativity in AUT but not TTCT was also found in a study on the effect of transcranial direct current stimulation (tDCS) on creativity [49]. The authors attributed the effect to release from inhibition. Interestingly, similar to our results, this study also found enhancement in the originality aspect of creativity, but not in fluency or flexibility. This may further imply that the placebo subjects in the present study were similarly less inhibited. Limitations of the present study include a between-subjects design which cannot measure the ability of placebo to increase creativity within an individual. A benefit of studying a placebo effect on healthy individuals is that unlike the clinic, it is possible to employ both conditions on the same person to control for intra-subject variability. In this study we decided not to use this option because performing the CFG twice may create a learning curve or a habituation effect we wanted to avoid. In addition, although the order of the AUT and TTCT was counterbalanced, the CFG was always completed first. This ordering effect might have contributed to the significant findings on the CFG, given that participants completed it first and could have been more alert at the beginning of the experiment. The approaching significant findings on the AUT could be due to insufficient power. Future work can use the finding that AUT and CGF both pick up on the effects of placebo, and employ CFG and AUT in a counterbalanced way before and after placebo in order to address this issue. The study is limited to a single culture and context, and future work can explore placebo on creativity in other cultures and contexts. Further research can explore the mechanisms by which placebo can enhance cognitive and creative abilities. Such exploration can include, in addition to tests to elucidate psychological mechanisms, also physiological and neurological measures of systematic and autonomous changes[66, 67]. Placebo for enhancing cognitive abilities such as creativity may thus be a research field with beneficial potential. BODY.SUPPORTING INFORMATION: S1 DatasetDataset of all subjects and their creativity scores in the three creativity tests.(XLSX)Click here for additional data file. S1 TableCorrelations of performance on creativity tests with odor ratings.Values in the upper table are Spearman correlation coefficients, values in the bottom table are the corresponding p values (not corrected).(XLSX)Click here for additional data file. S2 TableCorrelations within and across the three creativity tests.Values above the diagonal represent Spearman correlation coefficients, values below the diagonal represent corresponding p values (not corrected).(XLSX)Click here for additional data file.

TITLE: Randomized, Double-Blinded, Placebo-Controlled Trial of Fibrinogen Concentrate Supplementation After Complex Cardiac Surgery ABSTRACT.BACKGROUND: Postoperative bleeding after heart operations is still a common finding, leading to allogeneic blood products transfusion. Fibrinogen and coagulation factors deficiency are possible determinants of bleeding. The experimental hypothesis of this study is that a first-line fibrinogen supplementation avoids the need for fresh frozen plasma (FFP) and reduces the need for any kind of transfusions. ABSTRACT.METHODS AND RESULTS: This was a single-center, prospective, randomized, placebo-controlled, double-blinded study. One-hundred sixteen patients undergoing heart surgery with an expected cardiopulmonary bypass duration >90 minutes were admitted to the study. Patients in the treatment arm received fibrinogen concentrate after protamine administration; patients in the control arm received saline solution. In case of ongoing bleeding, patients in the treatment arm could receive prothrombin complex concentrates (PCCs) and those in the control arm saline solution. The primary endpoint was avoidance of any allogeneic blood product. Patients in the treatment arm had a significantly lower rate of any allogeneic blood products transfusion (odds ratio, 0.40; 95% confidence interval, 0.19 to 0.84, P=0.015). The total amount of packed red cells and FFP units transfused was significantly lower in the treatment arm. Postoperative bleeding was significantly (P=0.042) less in the treatment arm (median, 300 mL; interquartile range, 200 to 400 mL) than in the control arm (median, 355 mL; interquartile range, 250 to 600 mL). ABSTRACT.CONCLUSIONS: Fibrinogen concentrate limits postoperative bleeding after complex heart surgery, leading to a significant reduction in allogeneic blood products transfusions. No safety issues were raised. ABSTRACT.CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01471730. BODY: In complex cardiac surgery, operations with prolonged cardiopulmonary bypass (CPB), allogeneic blood products transfusions are often necessary to control perioperative bleeding and reverse excessive hemodilution. Fresh frozen plasma (FFP), platelet concentrates (PLT), and cryoprecipitate (CRYO) are the most commonly blood-derived procoagulant agents, and packed red cells (PRCs) are used to maintain hemoglobin levels. FFP is the most commonly prescribed hemostatic agent,1 with more than 300 000 units annually transfused in the UK2 and approximately 4.5 million units in the United States in 2011,3 without any sign of decrease over the last 10 years.4 At present, FFP can be replaced by separate substitutes (albumin, antithrombin, coagulation factors, fibrinogen concentrate, and so on), depending on the different clinical and therapeutic needs. In the setting of bleeding control in complex cardiac surgery, the main reason for using FFP should be bleeding control in case of inadequate concentration/activity of coagulation factors and/or fibrinogen. A low activity of many coagulation factors is detectable after complex cardiac surgery, even if it may not be associated with postoperative bleeding.5 Conversely, low levels of fibrinogen after cardiac surgery have been associated with increased postoperative bleeding in different studies.5–7 Using FFP to correct low values of coagulation factors and/or fibrinogen may represent an inefficient treatment, given that it has been shown that large doses (up to 2 L) of FFP are required especially to correct fibrinogen deficits.8,9 Fibrinogen levels may be corrected using CRYO, but again large doses are required, and the availability of CRYO is limited or absent in many countries.10 The present study is aimed to verify the hypothesis that a FFP-free strategy, based on a first-line fibrinogen concentrate supplementation and a (facultative) second-line prothrombin complex concentrate (PCC) supplementation, is effective in reducing any kind of allogeneic blood product transfusions after complex heart surgery. BODY.METHODS.STUDY DESIGN AND POPULATION: This is a prospective, randomized, double-blind, placebo-controlled, parallel-group, clinical study conducted at a single institution (IRCCS Policlinico San Donato, San Donato Milanese, Milan, Italy). The study was approved by the local ethics committee of Melegnano (Milan) ASL2 and by the Italian Agency of Drugs. The study was assigned the EudraCT code 2011-0046-33 and was registered at ClinicalTrials.gov with the identifier code NCT01471730. The study was spontaneous, with the external support of CSL Behring, which provided the study drugs (fibrinogen concentrate and PCCs) and the ROTEM® reagents free of charge. CSL Behring had no role in data collection, analysis, and quality control. Data property belongs to the IRCCS Policlinico San Donato. All patients signed a written informed consent to participate in the study. The study began on November 29, 2011. Eligible patients had to fulfill the following criteria: age 18 years or older; planned complex cardiac surgery operation with expected CPB duration of 90 minutes or longer; and presence of at least one of the following risk factors11: age >65 years; non-elective surgery; serum creatinine level >1.36 mg/dL; and redo surgery. Exclusion criteria were: participation in another randomized, controlled trial; refusal to participate; emergency surgery; known congenital or acquired coagulopathy; baseline antithrombin levels <80%; known autoimmune disorder; pregnancy; and major antiplatelet agents (P2Y12 inhibitors) not discontinuated in the 5 days before surgery. In patients with P2Y12 inhibitors discontinuated between 6 and 10 days before surgery, platelet function was tested and the patient was admitted to surgery (and to the study) according to predefined cut-off values as per our routine practice.12,13 Additional exclusion criteria were included to avoid the risk of transfusions triggered by preoperative anemia and/or excessive intraoperative hemodilution. These included a preoperative hematocrit (HCT) value <35% and a body surface area <1.7 m2. During surgery, withdrawal criteria were established again to avoid hemodilution-related transfusions. Intraoperative PRC transfusions before study drug administration and excessive hemodilution during CPB (HCT <23%) were withdrawal criteria. Patients screened and not withdrawn were randomized to the treatment arm or the control arm. BODY.METHODS.SURGERY AND CPB: All the patients received our standard surgical treatment and CPB technique. This included the use of reduced priming volume of the oxygenator and circuit, vacuum-assisted drainage, use of cell-saver intraoperatively, and use of postoperative systems to collect, process, and reinfuse chest drain blood. All patients received a total intraoperative dose of 30 mg/kg of tranexamic acid. CPB was established after a loading dose of 300 IU/kg of unfractionated heparin plus additional doses to reach and maintain a target activated clotting time of 450 seconds or longer. BODY.METHODS.INTERVENTIONS: Patients were randomly allocated to a treatment arm or a placebo (0.9% saline solution) arm. Twenty minutes before the removal of the aortic cross-clamp, patients were tested with a ROTEM® (TEM International, Munich, Germany) tissue-factor activated (EXTEM) and fibrin-based thromboelastometry test (FIBTEM). Clotting time (CT; seconds) at EXTEM and maximum clot firmness (MCF; mm) at the EXTEM and FIBTEM were recorded. After protamine administration (1:1 ratio heparin loading dose-protamine dose), patients in the treatment arm received fibrinogen concentrate (Haemocomplettan®; CSL Behring, Marburg, Germany) at a dose that depended on the MCF at the FIBTEM, according to the previously published equation14,15:14,15 After 15 minutes from fibrinogen concentrate (or placebo) administration and in the presence of ongoing microvascular bleeding, a second EXTEM was performed, and in case of a CT longer than 80 seconds, the patients of the study arm were planned to receive 4-factors PCCs (Confidex®; CSL Behring, Marburg, Germany) at a dose of 7 U/kg, whereas patients in the control arm received placebo treatment. In the presence of ongoing microvascular bleeding intra- or postoperatively, bleeding and anemia control was guaranteed by our standard protocol recently published in a modified version,16 which considers: FFP if the international normalized ratio (INR) >1.5 or the reaction time at thromboelastography (Haemoscope, Niles, IL) in a heparinase cup (to rule out residual heparin effects) >12 minutes. Platelet concentrate in the case of a platelet count <50 000 cells/μL. PRCs (1 unit at a time) under the following conditions: always if the hemoglobin (Hb) value <7 g/dL; possible without medical justification if Hb value between 7 and 7.9 g/dL; possible but with medical justification (hemodynamic instability; oxygen extraction rate >40%; signs of organ ischemia) if Hb value between 8 and 9 g/dL; never if Hb value >9 g/dL. In the presence of bleeding refractory to the above treatment, determining severe hemodynamic instability, and for all the life-saving conditions, an empirical rescue therapy was allowed, inclusive of the use PCCs, fibrinogen concentrate, and recombinant activated factor VII. Surgical revision was considered in the presence of ongoing bleeding without signs of coagulopathy at the viscoelastic and conventional laboratory tests. BODY.METHODS.MEASUREMENTS: Data collection was performed using our institutional database, which includes all the demographics, preoperative factors, procedure details, intraoperative data, and outcome measurements. Transfusion risk was assessed using a transfusion risk score,17 whereas the overall severity of the patients' profile was assessed using the logistic EuroSCORE II.18 For the purpose of the present study, the following additional data have been collected: FIBTEM MCF; EXTEM CT; fibrinogen values at the arrival in the intensive care unit (ICU; (Clauss method); and platelet count at the arrival in the ICU. BODY.METHODS.STUDY ENDPOINTS: The primary endpoint of this study was avoidance of any allogeneic blood products usage (inclusive of PRCs, FFP, and PLT). CRYO were not considered, given that it is not available at our institution. For the primary endpoint, the window of observation was hospital stay up to 30 days. Secondary endpoints were: number of PRC, FFP, and PLT units (same window of observation as for the primary endpoint); massive blood transfusion (7 units or more of PRCs in the first 24 postoperative hours); postoperative bleeding (chest drain output in the first 12 postoperative hours); and surgical revision as a result of bleeding (first 48 postoperative hours). Safety endpoints were operative mortality and thromboembolic complications (in-hospital or within 30 days from surgery after hospital discharge). BODY.METHODS.SAMPLE SIZE: Sample size was based on the primary endpoint (allogeneic blood products transfusions). Historical data from the last 5 years were retrieved from the institutional database by selection of the patients fulfilling the inclusion/exclusion/withdrawal criteria of the present study. Allogeneic blood products transfusion rate was 61%. Effect size was settled at a transfusion rate of 35% for the treatment arm and 60% for the control arm. With a 2-sided alpha value of 0.05 and beta value of 0.20, the sample size was calculated at 116 patients (58 in each arm). Considering a withdrawal rate of 3%, the number of patients to be enrolled was settled at 120. BODY.METHODS.RANDOMIZATION AND MASKING: A randomization code was generated with a computerized system. Sealed envelopes containing the randomization codes were prepared. The randomization ratio of the treatment arms was 1:1. The randomization process was managed by an unblinded biologist (E.B.). The unblinded biologist was in charge of drug or placebo preparation, which was performed in a laboratory annexed to the operating theater. The FIBTEM/EXTEM tests were done in the same laboratory by the same biologist. Drug or placebo were prepared in sterile bags after performing the FIBTEM/EXTEM tests. Each gram of fibrinogen concentrate was diluted in 50 mL of sterile water or an equivalent volume of 0.9% saline solution as placebo. The same procedure was followed for PCCs. Medication(s) were delivered to the operating room after protamine administration (fibrinogen concentrate or placebo) or at the request of the attending anesthesiologist in bleeding patients (PCCs or placebo). The attending anesthesiologist, the surgical staff, and doctors in charge for the clinical care in the ICU and ward were blinded, as well as the person in charge of the institutional database data entry. BODY.METHODS.STATISTICS: All data are expressed as number and percentage; continuous variables are expressed as mean with SD for normally distributed variables and as median with interquartile range (IQR) for non-normally distributed variables. Efficacy and safety analysis of between-arms differences were based on a Pearson chi-square for frequency differences, on a Student t test for differences in continuous, normally distributed variables, and on nonparametric tests (Mann-Whitney U test) for differences in continuous, non-normally distributed variables. Post-hoc analyses included linear or nonlinear regression models, aimed to determine the association between postoperative bleeding and coagulation tests and between treatment, transfusion risk, CPB time, and transfusions. Additional descriptive analyses on severely bleeding patients were performed. An intention-to-treat analysis was applied. All the statistical analyses were performed with a computerized package (SPSS 13.0; IBM, Chicago, IL). BODY.RESULTS.PATIENTS AND PROCEDURES: The general screening, enrollment, and randomization procedure is shown in Figure 1. The first patient was randomized on November 30, 2011 and the last on December 10, 2014. Three patients were withdrawn after enrollment: 1 patient did not receive surgery, and the other 2 were transfused before completion of CPB. All patients received the allocated treatment (fibrinogen or placebo); no patient in the treatment arm fulfilled the criteria for the receiving PCCs, and 4 patients in the placebo arm underwent the second treatment and received placebo. Figure 1Diagram showing the flow of participants through each stage of the trial. CPB indicates cardiopulmonary bypass; CT, clotting time; EXTEM, tissue-factor activated thromboelastometry test; PCCs, prothrombin complex concentrates. Adherence to the transfusion protocol was 100% in both arms. The final analysis for efficacy and safety include 58 patients in each arm. Baseline clinical and demographic characteristics of patients are shown in Table1. There was no significant imbalance between groups for any variable. Table 1 Baseline Clinical and Demographic Characteristics Intervention Arm Control Arm P Value Age, y 72 (64 to 76) 73 (68 to 79) 0.128 Weight, kg 74.5 (13.4) 74.7 (15.3) 0.939 Body surface area, m 2 1.85 (0.19) 1.86 (0.20) 0.808 Gender male 41 (71) 43 (74) 0.678 Left ventricular ejection fraction, % 57 (50 to 60) 57 (45 to 63) 0.600 Congestive heart failure 6 (10) 10 (17) 0.281 Active endocarditis 1 (2) 1 (2) 1.000 Serum creatinine, mg/dL 1.0 (0.8 to 1.1) 1.05 (0.8 to 1.2) 0.199 Serum bilirubin, mg/dL 0.5 (0.5 to 0.8) 0.5 (0.5 to 0.7) 0.810 Hematocrit, % 40.3 (3.2) 40.0 (2.9) 0.577 Chronic obstructive pulmonary disease 4 (7) 7 (12) 0.342 Previous cerebrovascular accident 2 (3) 4 (7) 0.402 Diabetes on medication 4 (7) 10 (17) 0.087 Redo surgery 12 (21) 8 (14) 0.326 Non-elective surgery 2 (3) 4 (7) 0.402 Transfusion risk score 15 (10 to 17) 15 (13 to 17) 0.278 EuroSCORE II 3.7 (2 to 5.6) 4.7 (2 to 8.4) 0.196 Type of surgery  CABG+mitral valve 8 (14) 9 (15) 0.793  CABG+aortic valve 13 (22) 13 (22) 1.000  CABG+LV aneurysmectomy 4 (7) 5 (9) 0.729  Double/triple valve 9 (15) 8 (14) 0.793  Ascending aorta 12 (21) 16 (28) 0.385  Others 12 (21) 7 (12) 0.210 Cardiopulmonary bypass time, min 105 (39) 111 (40) 0.367 Lowest hematocrit on CPB 28.5 (3.3) 27.8 (3.5) 0.294 Total heparin dose, IU×100 245 (210 to 300) 240 (200 to 271) 0.424 Total protamine dose, mg 250 (200 to 300) 245 (200 to 280) 0.973 Data are number (percentage) or median (interquartile range) or mean (SD). CABG indicates coronary artery bypass graft; CPB, cardiopulmonary bypass; IU, international units; LV, left ventricle. Based on the values of the FIBTEM MCF measured 20 minutes before aortic cross-clamp removal and the equation above reported, patients in the treatment arm received fibrinogen concentrate at a median dose of 4 g (IQR, 3 to 6). BODY.RESULTS.COAGULATION-RELATED LABORATORY DATA: Table2 reports coagulation-related laboratory data, as measured during the procedure (ROTEM) and at arrival in the ICU. After dosing, there was a significant (P=0.001) shortening of clotting time at the EXTEM in the treatment arm and no changes in the control arm. This led to a significant between-groups difference. Patients in the treatment arm demonstrated a significant increase in clot firmness both at EXTEM and FIBTEM, whereas patients in the control arm did not. At the arrival in the ICU, patients in the treatment arm had significantly higher levels of fibrinogen, as measured by the central laboratory. Table 2 Coagulation Data During the Procedure and at the Arrival in the ICU Parameter Treatment Arm Control Arm Mean Difference (95% CI) P Value Clotting time EXTEM—seconds, median (IQR)  20 minutes before removal of aortic cross clamp 84 (75 to 116) 89 (79 to 116) −4.6 (−24 to 15) 0.262  After dosing 78 (63 to 111) 95 (76 to 113) −16 (−36 to 3.7) 0.011 Maximum clot firmness EXTEM—mm, median (IQR)  20 minutes before removal of aortic cross clamp 59 (55 to 64) 59 (54 to 62) 1.7 (−0.6 to 4) 0.234  After dosing 64 (61 to 67) 60 (51 to 62) 7 (4.8 to 9.2) 0.001 Maximum clot firmness FIBTEM—mm, median (IQR)  20 minutes before removal of aortic cross clamp 13 (9 to 16) 13 (10 to 15) 0.3 (0.2 to 1.7) 0.705  After dosing 23 (21 to 25) 13 (9 to 15) 10 (9.1 to 11.6) 0.001 Fibrinogen (mg/dL)—median (IQR)  At the arrival in the ICU 367 (329 to 410) 242 (199 to 300) 120 (94 to 144) 0.001 Platelet count (cells, ×1000/μL)—median (IQR)  At the arrival in the ICU 126 (108 to 158) 124 (96 to 150) 9.5 (−14 to 24) 0.321 CI indicates confidence interval; EXTEM, tissue-factor activated thromboelastometry test; FIBTEM, fibrin-based thromboelastometry test; ICU, intensive care unit; IQR, interquartile range. BODY.RESULTS.EFFICACY ENDPOINTS: Primary and secondary efficacy endpoints are reported in Table3. During hospital stay, 39 (67.2%) patients in the treatment arm were free from any allogeneic blood product transfusions, a significantly (P=0.012) higher rate than those in the control arm (44.8%). The absolute difference in transfusion avoidance was 13 (22.4%) patients (relative difference for transfusion avoidance, 1.6; 95% confidence interval [CI], 1.07 to 2.42). Table 3 Efficacy Endpoints Endpoint Treatment Arm Control Arm OR (95% CI) P Value Primary endpoint  Avoidance of allogeneic blood products   Any product—no. of patients (%) 39 (67.2) 26 (44.8) 0.40 (0.19 to 0.84) 0.015   Packed red cells—no. of patients (%) 39 (67.2) 26 (44.8) 0.40 (0.19 to 0.84) 0.015   Fresh frozen plasma—no. of patients (%) 58 (100) 50 (86.2) N/A 0.006   Platelet concentrates—no. of patients (%) 58 (100) 54 (93.1) N/A 0.119 Secondary endpoints  Transfusions (overall patient population)   Packed red cells—no. of units, median (IQR) 0 (0 to 1) 1 (0 to 2) N/A 0.010   Fresh frozen plasma—no. of units, median (IQR) 0 (0 to 0) 0 (0 to 0) N/A 0.002   Platelet concentrates—no. of units, median (IQR) 0 (0 to 0) 0 (0 to 0) N/A 0.023  Transfusions (only patients receiving transfusions)   Packed red cells—no units, median (IQR) 2 (1 to 2) 2 (1 to 4) N/A 0.224   Postoperative bleeding—mL/12 h, median (IQR) 300 (200 to 400) 355 (250 to 600) N/A 0.042  Massive red blood cells transfusion 0 (0) 2 (3.4) N/A 0.496  Surgical revision 0 (0) 2 (3.4) N/A 0.496 CI indicates confidence interval; IQR, interquartile range; N/A, not applicable; OR, odds ratio. Among the secondary endpoints, postoperative bleeding, as recorded from chest drains during the first 12 postoperative hours, was significantly lower in the treatment arm, and the total amount of PRCs, FFP, and PLT used during hospital stay was significantly lower. When limited to patients receiving PRC transfusions, the difference in transfused units between the 2 arms was not statistically different. Figure 2 shows the total amount of allogeneic blood products transfused in the 2 study arms from the end of CPB through the whole postoperative hospital stay. Figure 2Total amount of allogeneic blood products transfused in the 2 study arms from the end of cardiopulmonary bypass to hospital discharge. BODY.RESULTS.SAFETY: Overall, the patient population experienced a morbidity/mortality rate that is compatible with the complexity of the surgical procedure. The overall mortality rate was 3.6%, lower than the predicted mortality rate according to the EuroSCORE II (5.6%; 95% CI, 4.5 to 6.7), with 1 (1.8%) event in the treatment arm and 3 (5.3%) in the control arm. The patient who died in the treatment arm had a postoperative low cardiac output refractory to drug therapy and intra-aortic balloon pump, developed a systemic blood infection with multiorgan failure, received a tracheostomy, and finally died after 34 days postsurgery. Postoperative morbidity was observed in 34 (60.7%) of the patients in the treatment arm and 36 (64.3%) in the control arm. Adverse events observed were typical for patients undergoing this kind of surgery, without between-groups differences. Thromboembolic complications were specifically investigated and are presented in Table4. There were no differences between groups; none of the adverse events observed was considered related to the study drug(s). No acute reaction to study drug(s) administration was observed. Table 4 Safety Endpoints Event Treatment Arm Control Arm P Value Any kind of complication 35 (60.3) 38 (65.5) 0.564 Low cardiac output 9 (15.5) 14 (24.1) 0.244 Atrial fibrillation 24 (41.4) 24 (41.4) 1.000 Lung dysfunction 1 (1.7) 0 (0) 0.315 Acute kidney injury 5 (8.6) 6 (10.3) 0.751 Infections  Bloodstream 3 (5.2) 4 (6.9) 0.697  Pneumonia 0 (0) 0 (0) 1.000  Urinary tract 4 (6.9) 7 (12.1) 0.342  Wound 0 (0) 0 (0) 1.000  Mediastinitis 0 (0) 0 (0) 1.000 Thromboembolic events  Myocardial infarction 0 (0) 0 (0) 1.000  Stroke 0 (0) 0 (0) 1.000  Peripheral thromboembolism 0 (0) 0 (0) 1.000  Pulmonary thromboembolism 0 (0) 0 (0) 1.000  Mesenteric infarction 0 (0) 1 (1.7) 0.315 Operative mortality 1 (1.7) 3 (5.2) 0.309 Data are number (%). BODY.RESULTS.POST-HOC ANALYSES: Patients with severe postoperative bleeding were identified according to the Universal Definition of Perioperative Bleeding19 as those who had a postoperative chest drain loss >1000 mL/12 h. These were 5 patients: 4 in the control arm and 1 in the treatment arm. Preoperative standard coagulation values were in the normal range in all these patients, with an aPTT between 31.3 and 35 seconds, an INR between 1.07 and 1.16, and PLT count between 150 000 and 212 000 cells/μL. The preoperative HCT ranged from 35% to 39.3%. No patient was under warfarin or oral anticoagulants. Two patients were redo cases; 3 patients received ascending aorta surgery, 1 coronary artery bypass graft (CABG)+mitral valve repair, and 1 mitral valve and aortic valve replacement. Only 1 patient required surgical revision. Two patients in the control arm were treated with PCCs on top of FFP and PLT concentrates. After correction for transfusion risk score and CPB duration, the treatment arm remained independently associated with transfusion avoidance in a multivariable logistic regression analysis, with an odds ratio (OR) of 0.43 (95% CI, 0.19 to 0.98, P=0.046). For a fixed CPB duration of 90 minutes, the absolute difference in transfusion risk at 4 different levels of transfusion risk score (5, 10, 15, and 20 points) was 6%, 12%, 19%, and 21%, respectively. A sensitivity analysis aimed to determine the association between postdosing viscoelastic tests/fibrinogen values and chest drain output in the first 12 postoperative hours was applied. Linear and nonlinear regression analyses were performed, and the best fit was found for a power equation (Figure 3). There was a significant (P=0.001) negative association between fibrinogen values at arrival in the ICU and postoperative bleeding. Similarly, there was a significant (P=0.008) negative association between MCF at FIBTEM and postoperative bleeding. The negative relationship between fibrinogen levels at arrival in the ICU and postoperative bleeding remained significant (P=0.05) after adjustment for the treatment arm. Figure 3Fibrinogen levels and postoperative bleeding. Dashed lines are 95% confidence interval. ICU indicates intensive care unit. No association was found between CT at EXTEM and postoperative bleeding. BODY.DISCUSSION: The results of the present study can be summarized as follows: (1) In the setting of complex cardiac surgery with CPB, FFP transfusions could be completely avoided using a strategy based on purified concentrates of coagulation factors; (2) within this strategy, fibrinogen alone was needed to control postoperative bleeding, without any patient requiring PCCs; and (3) this approach resulted in a significantly lower need for PRC transfusions, lower amount of allogeneic blood products transfused, and lower postoperative bleeding. Our study confirms the findings of previous small, randomized, controlled trials and prospective studies where an early use of fibrinogen concentrate was associated with a bleeding containment and less allogeneic blood products (especially FFP) use.20–22 Of notice, no patient in the treatment arm required PCCs. Our zero-plasma protocol was based on a first-line supplementation with fibrinogen concentrate, and only as a second step the patients would have received PCCs, in case of ongoing bleeding and prolonged clotting time at EXTEM. This condition was never observed in treatment arm patients and rarely in control arm patients. A deficiency of coagulation factors (excluding fibrinogen and factor XIII) leads to a decreased thrombin generation with prolonged CT at EXTEM, whereas a deficiency in fibrinogen leads to decreased clot firmness with reduced MCF at EXTEM and FIBTEM. Previous studies have highlighted that this second mechanisms is associated with postoperative bleeding in cardiac surgery, whereas the first is not.5 Our data support this interpretation, with 3 additional data emerging from the viscoelastic and conventional tests before and after fibrinogen administration: (1) Fibrinogen replenishment actually not only increases clot firmness, but also even reduces CT; (2) no association exists between CT and postoperative bleeding; and (3) there is a significant association between fibrinogen levels at arrival in the ICU and postoperative bleeding. Of notice, patients in the control arm rarely experienced very low postoperative fibrinogen levels (only 1 patient <150 mg/100 mL and 11 patients <200 mg/100 mL). Despite this, raising fibrinogen levels with fibrinogen concentrate was effective in reducing chest drain blood loss. This result was obtained without reaching abnormally high fibrinogen levels (only 3 patients between 500 and 550 mg/100 mL). Our results were confirmative of an association between postoperative fibrinogen levels and bleeding tendency. However, the relationship was not linear, and within a wide range of postoperative fibrinogen values (between 280 and 550 mg/dL), there was no association; below the value of 280 mg/dL, the relationship acquired significance and all the severely bleeding patients belonged to this region. According to a post-hoc analysis, the efficacy of prophylactic fibrinogen supplementation was more evident in patients at higher bleeding risk, with an absolute reduction in transfusion rate that reached 21% when the transfusion risk score exceeded 20 points. The present study is a "proof of concept" that replacing FFP with fibrinogen concentrate is an effective strategy for avoiding allogeneic blood product transfusion in complex heart surgery; however, it is not intended to suggest that all patients undergoing complex heart surgery should be prophylactically treated with fibrinogen at the end of CPB. The combination of complex surgery with a high transfusion risk score is probably the best environment to consider this option. Postoperative bleeding and transfusion needs are both multifactorial and complex items in cardiac surgery. Postoperative bleeding may result from a number of factors, including low PLT count or PLT function defects, deficiency of fibrinogen and soluble coagulation factors, and residual effects of heparin, hyperfibrinolysis, and surgical sources.23 Studies dealing with prohemostatic agents having postoperative bleeding/transfusions as the primary endpoint must deal with this multifactorial mechanism underlying bleeding during and after cardiac surgery and should minimize the effects of confounders. In our protocol, we have applied a number of procedures to minimize the effects of potential confounders, as well as to better stress the effects of the study variable (first-line use of fibrinogen concentrate and only second-line use of PCCs to replace FFP). Patients were operated on over a 3-year period by the same 4 surgeons, thus minimizing surgeon-related variability in bleeding control. Additionally, a patient blood management program has been established in our institution for many years, based on preoperative assessment of PLT function, intraoperative minimization of hemodilution, postoperative use of point-of-care coagulation tests (thromboelastography), and the application of a strict transfusion protocol. Overall, the patients in this study have been treated according to our usual policy of patient blood management, therefore following the philosophy of a pragmatic randomized, controlled trial. Within our strategy, a number of measures minimized the effects of possible confounders. Decision making on the timing of surgery in patients under double anti-PLT therapy was based on PLT function tests, rather than on an arbitrary discontinuation time.12,13 All patients received tranexamic acid to control hyperfibrinolysis. Presence of residual heparin effects after protamine administration was ruled out with specific tests with and without heparinase. Transfusion of allogeneic blood products is strongly institution dependent, as demonstrated by the high intercenter variability in a recent randomized, controlled trial, where only the hospital where surgery took place was a predictor of transfusion rate.24 This particularly applies to red blood cell transfusions, which may be triggered by anemia and hemodilution rather than by bleeding. To this respect, we tried to limit the potential confounding effect of these factors, excluding or withdrawing patients at high risk for receiving PRC transfusion as a result of non-bleeding-related anemia.24 Moreover, the standard application of a well-established transfusion protocol (without protocol violations) strongly reduced the attending physician-related variability in the allogeneic blood product transfusions. Overall, our reduction of the "background noise" that is quite typical in transfusion and bleeding-based studies probably created the adequate environment for assessing the efficacy of the zero-plasma strategy, leading to the achievement of the primary endpoint. Among the secondary endpoints, we believe that the evidence of a postoperative bleeding containment associated with the zero-plasma strategy is of particular relevance. The potentially beneficial effects of replacing FFP with fibrinogen concentrate rely on a better control of bleeding tendency, and no direct effect of this strategy in limiting allogeneic blood products transfusions could be advocated in the absence of a significant decrease in blood loss. From the safety point of view, no concerns were raised with respect to possible adverse effects related to fibrinogen supplementation, with no significant between-groups differences in morbidity events. The administration of prohemostatic drugs in the setting of cardiac surgery is always burdened by the potential risk of thromboembolic complications, as reported with the use of activated25 and nonactivated26 coagulation factors. Our study confirms previous reports about the safety of fibrinogen concentrate supplementation. Our series of treated patients reached values of MCF at the EXTEM that never exceeded the upper limit of the reference range,27 with an increased contribution of fibrinogen compensating for the reduced post-CPB PLT count and function. However, no patient in our study actually received both fibrinogen concentrate and PCCs. Therefore, we cannot exclude that this combination of factor concentrates may actually result in a greater risk of thromboembolic complications. Our study has a number of limitations. The first is its single-center protocol, which may be seen both as a strength (by reducing experimental variability) or a weakness (the zero-plasma strategy may be seen as effective only if applied in a specific center with a number of additional strategies already in place). A second limitation is the exclusion of patients with anemia and small body surface area; albeit justified by the need to avoid confounding factors determining non-bleeding-related transfusions, this patient selection limits the generalizability of our findings. A third point pertains the timing of fibrinogen supplementation. Our protocol is based on a first-line, pre-emptive strategy of fibrinogen replenishment in patients at high risk for postoperative bleeding, before bleeding takes place. It is not the intention of the present study to suggest routine fibrinogen administration in this subgroup of patients: We decided to follow a prophylactic strategy to investigate the efficacy of a zero-plasma approach in a controlled environment and not to wait until excessive bleeding was diagnosed. Studies based on a postoperative bleeding trigger for testing the efficacy of prohemostatic drugs are carried by a screening failure rate >80%25 that is unacceptable in a single-center trial. It is our feeling and clinical practice not to start treating postoperative bleeding before it appears clinically relevant (even if this may delay the onset of treatment leading to less beneficial effects), nor to treat numbers rather than bleeding. The suggestion from our study is that in the presence of excessive bleeding after heparin neutralization, associated with low fibrinogen levels, fibrinogen supplementation may be an adequate choice, avoiding the need for PCCs and/or FFP. An exact cut-off point for fibrinogen levels triggering fibrinogen supplementation is still to be determined, and its finding is outside the study purposes. Larger patient populations are required to determine at which level of functional (FIBTEM) fibrinogen or conventional (Clauss) fibrinogen value fibrinogen supplementation is required. Another point of debate is the adequate fibrinogen concentrate dose to achieve a clinically relevant effect. Our protocol is based on data from previous studies14,15 where a target MCF value of 22 mm at FIBTEM was proposed. This led to a median dose of 4 g of fibrinogen concentrate supplementation, which is more than the dose that we usually consider (2 g), but, however, much less than the dose reported in other studies19 (8 g). The dose used in our study and the target value of 22 mm for the MCF at FIBTEM have been effective in reducing bleeding and postoperative transfusion needs, avoiding the use of PCCs and FFP. However, it is possible that lower targets of MCF, as well as lower doses of fibrinogen concentrate, may be effective as well. In conclusion, our study demonstrates the efficacy of a zero-plasma strategy in complex cardiac surgery, based on a first-line treatment with fibrinogen concentrate. No safety issues related to this strategy were raised. Open questions remain with respect to the cost benefit of this approach, trigger values of fibrinogen concentration, and fibrinogen concentrate dose to be used. BODY.APPENDIX: The Surgical and Clinical Outcome Research (SCORE) group includes the following members: Marco Ranucci, MD, FESC, San Donato Milanese (Milan); Alessandro Frigiola, MD, San Donato Milanese (Milan); Lorenzo Menicanti, MD, San Donato Milanese (Milan); Serenella Castelvecchio, MD, FESC, San Donato Milanese (Milan); Ekaterina Baryshnikova, PhD (Biol), San Donato Milanese (Milan); Valeria Pistuddi, San Donato Milanese (Milan); Santi Trimarchi, MD, San Donato Milanese (Milan); Concetta Carlucci, MD, San Donato Milanese (Milan); Alessandro Varrica, MD, San Donato Milanese (Milan); Angela Satriano, MD, San Donato Milanese (Milan); Maria Teresa La Rovere, MD, FESC, Montescano (Pavia).

TITLE: Handwriting training in Parkinson’s disease: A trade-off between size, speed and fluency ABSTRACT.BACKGROUND: In previous work, we found that intensive amplitude training successfully improved micrographia in Parkinson's disease (PD). Handwriting abnormalities in PD also express themselves in stroke duration and writing fluency. It is currently unknown whether training changes these dysgraphic features. ABSTRACT.OBJECTIVE: To determine the differential effects of amplitude training on various hallmarks of handwriting abnormalities in PD. ABSTRACT.METHODS: We randomized 38 right-handed subjects in early to mid-stage of PD into an experimental group (n = 18), receiving training focused at improving writing size during 30 minutes/day, five days/week for six weeks, and a placebo group (n = 20), receiving stretch and relaxation exercises at equal intensity. Writing skills were assessed using a touch-sensitive tablet pre- and post-training, and after a six-week retention period. Tests encompassed a transfer task, evaluating trained and untrained sequences, and an automatization task, comparing single- and dual-task handwriting. Outcome parameters were stroke duration (s), writing velocity (cm/s) and normalized jerk (i.e. fluency). ABSTRACT.RESULTS: In contrast to the reported positive effects of training on writing size, the current results showed increases in stroke duration and normalized jerk after amplitude training, which were absent in the placebo group. These increases remained after the six-week retention period. In contrast, velocity remained unchanged throughout the study. ABSTRACT.CONCLUSION: While intensive amplitude training is beneficial to improve writing size in PD, it comes at a cost as fluency and stroke duration deteriorated after training. The findings imply that PD patients can redistribute movement priorities after training within a compromised motor system. BODY.INTRODUCTION: Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the basal ganglia leading to a number of motor symptoms [1]. Aside from the primary motor symptoms, handwriting difficulties occur frequently and are generally known as micrographia, i.e. a reduction in writing amplitude [2]. These difficulties commonly result in reduced legibility. While micrographia is one of the important features of handwriting impairment in PD, it is not the only deficit [3]. Letanneux et al. showed that three other variables may also differ from healthy controls, i.e. stroke duration, velocity and fluency [3]. Altogether, smaller writing size, longer stroke duration, slower handwriting velocity and reduced fluency point to dysgraphic handwriting [4–9], which characterizes performance in PD better than micrographia alone. Treatment with dopaminergic medication or deep brain stimulation was found to have a beneficial effect on the latter three variables, but not on amplitude [10–13]. As such, there is an unmet need for rehabilitation interventions that target writing size, which can be considered as the most important parameter determining handwriting legibility. In a recent randomized controlled trial, our group showed solid improvements of writing amplitude after six weeks of intensive amplitude training, in addition to dopaminergic medication [14]. In healthy persons, amplitude and speed during voluntary arm and hand movements, such as writing, obey certain generally accepted rules of motor control: irrespective of the muscles involved, larger movement amplitudes are accompanied by an increase in velocity [15]. However, in patients with PD this speed-amplitude relation was shown to be altered and characterized by either a reduced movement speed to maintain amplitude or by dysregulation of movement size to the benefit of movement speed in comparison to healthy controls [7, 16–18], expressing the symptomatic effects of bradykinesia. Also, patients with PD modulated acceleration measures inefficiently as compared with controls, i.e. had a lower mean acceleration and smaller peak acceleration compared to controls, mainly when they were requested to write with large stroke sizes [7]. Early EMG studies provided a partial explanation for this difference. While healthy persons needed a single EMG burst to execute arm movements, patients with PD required multiple bursts, a pattern which was aggravated during large-amplitude movements [19–22]. Previous studies in PD using graphics tablets have also reported prominent problems in movement fluency (see Letanneux et al. for a review [3]). Writing fluency can be measured by the normalized jerk of a writing trajectory, which reflects the change in acceleration normalized for different stroke durations and sizes [6]. An increased normalized jerk during several handwriting tasks was found consistently in PD, reflecting a reduced capacity to coordinate the fingers and wrist [6]. Most studies in this field investigated the speed, amplitude and fluency of writing during single session experiments, whereby writing at different sizes, velocities and complexity was compared [3]. So far, it has not been investigated how training of handwriting, with a focus on improving writing amplitude, may differentially affect writing size, stroke duration, speed and fluency and the relation among these variables after a multi-session training. Gaining insight into the effects of training on these parameters is crucial to refine future rehabilitation interventions for handwriting problems in PD. BODY.METHODS.PARTICIPANTS AND STUDY DESIGN: The same patient groups were included as described in an earlier paper by Nackaerts et al. [14], of which this study comprises a more detailed kinematic analysis. In short, 38 right-handed patients with PD participated [23]. Inclusion criteria were: (i) diagnosis of PD according to the United Kingdom PD Society Brain Bank criteria [24]; (ii) Hoehn and Yahr (H&Y) stage I to III in the on-phase of the medication cycle [25]; (iii) experiencing writing problems, as identified by a score of one or more on item 2.7 (Handwriting) of the Movement Disorder Society-sponsored revision of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS) part II [26]; and (iv) the absence of severe cognitive impairment (Mini-Mental State Examination (MMSE) ≥ 24) [27]. Exclusion criteria were: (i) color blindness or other impairments in vision interfering with handwriting; (ii) upper limb problems unrelated to PD; and (iii) deep brain stimulation. Patients were assigned to one of two training programs by means of a stratified randomization procedure based on H&Y stage and age. Eighteen patients were assigned to an intensive writing amplitude training (= EXP group) and 20 to a placebo group, who received a stretch and relaxation program designed not to influence writing amplitude (= PLB group). Both programs were equally time-intensive and included 30 minutes of practice, five days per week over the course of six weeks. All patients filled out a diary so that the amount and duration of the intervention could be closely monitored. As well, patients in both groups received a weekly follow-up by the researcher, during which feedback and support were provided. In short, writing training consisted of pen-and-paper exercises and exercises on the touch-sensitive tablet and was aimed at improving writing amplitude with the help of visual cues. The stretch and relaxation program aimed to teach patients how to alleviate tension in the upper limbs. More detailed information on general characteristics of both groups and the content of the training programs can be found in Nackaerts et al. [14]. The study design and protocol were approved by the local Ethics Committee of the KU Leuven and were in accordance with the code of Ethics of the World Medical Association (Declaration of Helsinki, 1967). After explanation of the study protocol written informed consent was obtained from all participants prior to participation in the study. The trial was registered as ClinicalTrials.gov Protocol Record G.0906.11. BODY.METHODS.OUTCOME MEASURES: The effect of training on stroke duration (s), writing velocity (cm/s) and normalized jerk was measured on a touch-sensitive tablet (sampling frequency 200 Hz, spatial resolution 32.5 μm) before and after the training program and after a six-week retention period. During all tests, two types of tasks were performed, i.e. an automatization and transfer task [14]. In short, for the automatization task patients were asked to write a 3-loop sequence at two sizes, 0.6 or 1.0 cm indicated by visual target zones, and this either alone or while counting high or low tones [28]. The transfer task incorporated writing of a trained and untrained sequence in the presence and absence of the visual target lines, resulting in four conditions. The trained sequence consisted of the same 3-loop sequence mentioned above. The untrained sequence involved a continuous figure 8-like movement [14]. Both trained and untrained sequences were performed with and without cues and at the two sizes described above. For both the automatization and transfer task, three blocks were performed in which each condition was provided in a random order. Each writing condition lasted 27 s and was followed by a rest period of 6 s. Participants were tested during the on-phase of the medication cycle at baseline, after six weeks of training (post-test) and after six weeks without training (retention-test). Medication intake was monitored and kept constant throughout the study. BODY.METHODS.DATA PROCESSING AND STATISTICAL ANALYSIS: All data from the tablet were filtered at 7 Hz with a 4th-order Butterworth filter and further processed using Matlab R2011b. Stroke duration (s), writing velocity (cm/s) and normalized jerk were extracted as dependent variables. The size and duration of individual up- and downstrokes were defined by calculating local minima and maxima. Writing velocity (cm/s) was calculated based on the duration and size of the individual strokes. The normalized jerk was calculated as the change in acceleration, normalized for different stroke durations and sizes making in a unit-free measure, using the formula described by Teulings et al. [6]: Normalizedjerk=12∫dtj2(t)×duration5length2, where j(t) is the third time derivative of position. Statistical analysis was performed using SPSS software (version 24). Both the automatization and transfer task were analyzed using a mixed model approach. For the automatization task Group (EXP or PLB), Time (baseline, post-training or retention) and Task (single or dual) were used as fixed factors and MAM-16 as a covariate, as it differed between both patient groups. To study transfer, Group, Time, Task (trained or untrained) and Cue (cued or uncued) were incorporated as fixed factors, with MAM-16 as a covariate. Both models controlled for the within-subject differences by including random effects for participants. Finally, partial correlation analyses, corrected for MAM-16, were performed between writing parameters at baseline and clinical characteristics, i.e. disease duration, cognition (MMSE) and disease severity (MDS-UPDRS total score and UL tremor and sequence subscores). Similarly, the difference between baseline and post-training and between baseline and retention for the writing parameters were correlated to the clinical characteristics for the EXP and PLB group separately. Significance levels for all tests were set at p < 0.05. BODY.RESULTS: Both amplitude conditions showed comparable results. Therefore, only results from the large-amplitude condition are described in detail. For results on the small-amplitude condition we refer the reader to the Supplementary results (S1 File), unless indicated otherwise. BODY.RESULTS.AUTOMATIZATION TASK.DURATION: For writing duration, a significant Group x Time interaction was found in the large-amplitude condition (F = 11.434, p < 0.001) (Fig 1A). Post hoc analysis showed that the EXP group had a longer stroke duration compared to the PLB group post-training (p = 0.004) and at retention (p = 0.015). In the EXP group there was an increase in duration from baseline to post-training (p < 0.001) and retention (p = 0.023), although duration decreased again from post-training to retention (p = 0.004). A significant decrease in duration from baseline to retention (p = 0.003) was found in the PLB group. 10.1371/journal.pone.0190223.g001Fig 1Automatization task in the large-amplitude condition.Mean and standard errors are displayed, corrected for MAM-16. (*) p < 0.1, * p < 0.05, ** p < 0.01, *** p < 0.001. BODY.RESULTS.AUTOMATIZATION TASK.VELOCITY: Also for velocity, the large-amplitude condition revealed a Time x Group interaction (F = 4.810, p = 0.011) (Fig 1B). Post hoc analysis showed no changes from baseline to post-training and an increase in velocity from post-training to retention in the EXP group (p = 0.002). On the other hand there was an increase in the PLB group from baseline to post-training (p = 0.011) and retention (p < 0.001) and from post-training to retention (p = 0.010). BODY.RESULTS.AUTOMATIZATION TASK.NORMALIZED JERK: A significant Time x Group interaction was found for the normalized jerk in the large-amplitude condition (F = 8.928, p < 0.001) (Fig 1C). Further analysis revealed a higher normalized jerk in the EXP group compared to PLB group post-training (p = 0.027). Additionally, the normalized jerk increased from baseline to post-training (p < 0.001) and retention (p = 0.054) in the EXP group, although there was a decrease again from post-training to retention (p = 0.005). In the PLB group on the other hand, the normalized jerk tended to decrease from baseline to retention (p = 0.069). A similar, though weaker pattern was found for the small amplitude (S1 File). BODY.RESULTS.AUTOMATIZATION TASK.CORRELATION ANALYSIS: Correlation analysis revealed that, at baseline, a higher normalized jerk correlated with more difficulties on the upper limb sequence items of the MDS-UPDRS-III (r = 0.391, p = 0.017). When looking at the effects of training, it was found that a greater increase in the normalized jerk correlated with a lower MMSE score (post-training: r = -0.734, p = 0.001; retention: r = -0.676, p = 0.003) (Fig 2A) and with more difficulties on the upper limb sequence items of the MDS-UPDRS-III (post-training: r = 0.462, p = 0.062; retention: r = 0.579; p = 0.015) for the EXP group only. Additionally, in the EXP group a greater increase in duration was correlated to more cognitive difficulties (post-training: r = -0.732, p = 0.001; retention: r = -0.624, p = 0.007) (Fig 2B). No correlations were found between the outcomes and the MDS-UPDRS-III upper limb tremor items. 10.1371/journal.pone.0190223.g002Fig 2Correlation between the difference from baseline to post-training on the automatization task and cognition.(A) A greater difference in normalized jerk correlates with a lower MMSE score in the experimental group; (B) A greater difference in movement duration correlates with a lower MMSE score in the experimental group. Represented values are not corrected for MAM-16. As the correlations with MMSE in the EXP group might have been driven by outliers, we performed a sensitivity analysis excluding the two outliers. This resulted in similar outcomes (S1 File). BODY.RESULTS.TRANSFER TASK.DURATION: A significant Time x Group interaction was apparent in the large-amplitude condition (F = 18.237, p < 0.001) (Fig 3A), indicating a longer duration in the EXP compared to PLB group post-training (p = 0.001) and at retention (p = 0.002) at post hoc analysis. These differences were driven by an increase in duration in the EXP group from baseline to post-training (p < 0.001) and to retention (p = 0.023), in combination with a decrease in duration in the PLB group from baseline to post-training (p = 0.007) and to retention (p < 0.001). 10.1371/journal.pone.0190223.g003Fig 3Transfer task in the large-amplitude condition.Mean and standard errors are displayed, corrected for MAM-16. (*) p < 0.1, * p < 0.05, ** p < 0.01, *** p < 0.001. BODY.RESULTS.TRANSFER TASK.VELOCITY: A significant Time x Group interaction was found for the large-amplitude condition (F = 13.888, p < 0.001), revealing a significant increase from baseline to post-training and retention in the PLB group (both p < 0.001) (Fig 3B). For the small-amplitude, post hoc analysis exposed an additional decrease in velocity from baseline to post-training in the EXP group (p = 0.036) (S1 File). Finally, in the large-amplitude condition a Group x Cue interaction was found (F = 4.572, p = 0.033), indicating that patients in the PLB group wrote faster in the cued compared to uncued condition (p = 0.002). BODY.RESULTS.TRANSFER TASK.NORMALIZED JERK: In line with the automatization task, the transfer task also showed a significant Time x Group interaction for the large-amplitude condition (F = 8.732, p < 0.001) (Fig 3C). There was a higher normalized jerk in the EXP compared to PLB group post-training (p = 0.012) and at retention (p = 0.052). In addition, there was an increase in normalized jerk of the EXP group from baseline to post-training (p = 0.001). Also, there was a tendency to increase normalized jerk in the EXP group (p = 0.078) and decrease the normalized jerk in the PLB group (p = 0.066) from baseline to retention. A similar, though weaker pattern was found for the small amplitude (S1 File). BODY.RESULTS.TRANSFER TASK.CORRELATION ANALYSIS: Correlation analysis showed that a higher normalized jerk was associated with more difficulties on the upper limb sequence items of the MDS-UPDRS-III (r = 0.346, p = 0.036), in line with results from the automatization task. Additionally, less fluent handwriting correlated with worse cognition (r = -0.324, p = 0.051). When looking at the effects of training, it was found that a greater increase in the normalized jerk correlated with a lower MMSE score (post-training: r = -0.550, p = 0.022) for the EXP group only. No correlations were found with the MDS-UPDRS-III upper limb tremor items. As the correlations with MMSE in the EXP group might have been driven by outliers, we performed a sensitivity analysis excluding the two outliers. This resulted in similar outcomes (S1 File). BODY.DISCUSSION: The present study revealed that, in addition to the previously detected improvement in writing size [14], other writing features are modified by intensive amplitude training. These adaptations included an increase in movement duration and a decrease in writing fluency across tasks and conditions, i.e. in both single and dual task, trained and untrained or cued and uncued sequences. On the contrary, the placebo group did not show beneficial effects on writing amplitude [14]. However, movement duration decreased, which led to an increase in writing velocity regardless of condition. Additionally, the placebo group showed an improvement of writing fluency in the automatization task. The training of amplitude is a common rehabilitation approach in patients with PD, which was originally applied to improve speech deficits [29]. This treatment concept, known as Lee Silverman Voice Treatment (LSVT®), underlies the more recently developed protocol called 'Training BIG', to improve movement amplitude in the limbs [30, 31]. During BIG, amplitude is chosen as the main focus of training to overcome bradykinesia/hypokinesia by a 'recalibration' of the patients' perception of normal amplitude execution. This amplitude focus is combined with an increase of intensity and complexity during training BIG, which is similar to the writing protocol used in this study. Interestingly, previous BIG trials showed positive effects on both the speed and amplitude during reaching movements, balance and bed mobility [30, 32, 33], except for one study on gait. In the latter, only stride length increased after training without increases in velocity or cadence in the as-fast-as-possible condition, confirming the possibility of a ceiling effect [30]. Importantly, none of the BIG trials analyzed the possible cost of amplitude-based training on motor fluency, cognitive load and energy consumption. Also, BIG training effects were largely studied on the trained tasks only, while in this study we analyzed trade-off and learning effects during automatization and transfer tasks. Finally, the differences between the training BIG studies and the current study could be a result of the task, as handwriting is a highly visually-driven movement incorporating both habitual and goal-directed motor control [34, 35]. We will interpret these results in the light of a proposed framework of movement parameter trade-off presented in Fig 4. Though not assessed in this study, the literature suggests that in a healthy system simply changing one movement parameter in one direction has an effect on the other parameters [15] (Fig 4, left panel). For instance, in healthy persons, movement speed was found to increase with rising amplitude [16, 36]. In contrast, the findings of the current and other studies on handwriting demonstrate that in patients with PD, velocity was found to saturate when amplitude increases (Fig 4, middle panel) [37–39]. The lack of concomitant increase in velocity (or velocity saturation) after amplitude training may be explained by a motor control impairment, affecting the speed-amplitude relation in patients with PD (Fig 4, middle panel). As a result, motor learning may come at a cost once patients reach the upper limits of their motor control system, explaining the stagnation in velocity. This view is supported by several studies showing that although patients with PD can alter one movement parameter, this occurs at the expense of another [4, 7, 40, 41]. 10.1371/journal.pone.0190223.g004Fig 4Movement parameter trade-off in health and disease.Dotted arrows indicate the possible interaction between disease-related changes and compensatory change in priorities. A second explanation for the deterioration of movement duration is in line with the cognitive reserve theory, i.e. the recruitment of cognitive or other compensatory processes to substitute for ageing or pathology (for a review see Stern [42]) due to a priority change (Fig 4, right panel). This priority shift may also explain the increase of the normalized jerk accompanying the benefits on writing size, which may be a direct consequence of a system overload expressed as a loss of movement fluency. Worsening of writing fluency when movements are executed more slowly or with a larger amplitude is in agreement with previous studies in healthy young adults and other patient groups [43, 44]. Interestingly, in support of this hypothesis, we found that a worse cognitive profile and more advanced disease, represented by more sequential upper limb difficulties, resulted in a greater trade-off for movement duration and normalized jerk. This implies that rather than a ceiling, the lack of change in velocity may have been the result of the compensatory strategy used, which impacted on fluency and speed. A final explanation, not illustrated in Fig 4, is that the placebo treatment consisting of stretching and relaxation was in fact beneficial, as the writing tasks were performed faster and with a decrease in the normalized jerk for the automatization task, although these changes were not necessarily beneficial for legibility of handwriting [45]. One specific explanation for the changes in velocity and fluency could be that the stretch and relaxation program influenced tremor positively. However, neither the correlation analysis with the upper limb tremor scores of the MDS-UPDRS-III, nor previous research on mindfulness training [46] supports this line of reasoning. Moreover, other rehabilitation studies showed that stretching and relaxation was not beneficial for motor performance in PD [47]. Therefore, we consider this explanation as unlikely. BODY.DISCUSSION.IMPLICATIONS FOR REHABILITATION: The most comprehensive interpretation of our results suggests that intensive amplitude training resulted in a trade-off between amplitude and movement duration and fluency. This implies that the compensatory strategy learned during training may come at a cost, which has consequences for clinical practice. This knowledge does not argue against motor learning or rehabilitation. Rather, it acknowledges that in patients with PD it is of utmost importance to select one single parameter which is crucial for the largest functional gains. When aiming to improve the legibility of handwriting, the main parameter to focus on seems to be movement amplitude. Progression of training could then be aimed to follow logical steps if the clinical profile of the patients allows it. One strategy could be to solely target the most important movement parameter during the entire program, as is currently done in BIG programs [30]. This approach is based on the hypothesis that the parkinsonian brain may solve kinematic challenges in separate steps. Improvements will mainly be found on the parameter to which main priority is given, inevitably coming at a cost regarding the secondary parameters. However, as training progresses, resources may gradually become available again and the deterioration of other variables will decrease, optimizing the functional outcome of the training program. We also found that movement duration decreased, velocity increased and fluency improved again in the experimental group from post-training to retention for the automatization task, in combination with a stable amplitude [14]. It is, however, important to note that these effects were detected after a period without practice. BODY.CONCLUSION: In conclusion, we found that intensive amplitude training, reported earlier to help writing amplitude, came at the expense of writing duration and fluency in patients with PD. Several hypotheses were put forward to explain the observed trade-off. While there likely is an interaction between the disease-related and the compensatory changes, our findings point towards a reorganization within the limited motor control system due to changing motor priorities. Further research is indicated to investigate whether a more extensive training program could address various aspects of handwriting comprehensively. Until then, we advise to focus on amplitude to improve legibility of handwriting in PD. BODY.SUPPORTING INFORMATION: S1 FileSupplementary results of the small-amplitude condition.(PDF)Click here for additional data file. S1 Dataset(XLSX)Click here for additional data file.

TITLE: Correct usage, ease of use, and preference of two dry powder inhalers in patients with COPD: analysis of five phase III, randomized trials ABSTRACT.BACKGROUND: Handheld inhalers are used to deliver treatment for COPD. Incorrect usage leads to suboptimal disease control. Complex treatment regimens and use of multiple inhalers may reduce patient compliance. The Anoro ElliptaTM dry powder inhaler (DPI) simultaneously delivers umeclidinium bromide (UMEC) and vilanterol (VI) without coformulation being required. ABSTRACT.AIM: To assess the correct usage and ease of use of the ElliptaTM DPI administering UMEC/VI and to compare patient preference for ElliptaTM with the HandiHaler® through exploratory analyses of patient and observer questionnaires in five Phase III studies. ABSTRACT.METHODS: Two Phase III, 3-month double-blind, placebo-controlled studies assessed the correct usage of the ElliptaTM DPI at Day 1 and after 6 weeks, and ease of use of the ElliptaTM DPI using a nonvalidated patient questionnaire after 6 weeks or early withdrawal. In three 6-month, blinded double-dummy, active comparator studies (two Phase IIIa and one Phase IIIb), patients completed a COPD device preference questionnaire between the ElliptaTM DPI and the Handi-Haler® at Day 168 (Week 24) or early withdrawal. ABSTRACT.RESULTS: In the 3-month placebo-controlled studies, ≥98% of patients used the ElliptaTM DPI correctly and 99% of patients found the inhaler easy/very easy-to-use and the dose counter easy/very easy to read. Across the two Phase IIIa active comparator studies, patients consistently stated a preference for the ElliptaTM DPI over HandiHaler® regarding the number of steps to use (59% vs 17%), time taken to use (62% vs 14%), and ease of use (63% vs 15%) regardless of which inhaler contained active drug. Results were consistent in the Phase IIIb active comparator study. ABSTRACT.CONCLUSION: Delivery of UMEC/VI via the ElliptaTM DPI was considered easy-to-use, and patients with COPD demonstrated clear preference for this inhaler compared with HandiHaler®. BODY.INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is a significant cause of morbidity and mortality, and its prevalence is predicted to rise with an aging population.1 Handheld inhalers are often used to treat COPD and deliver drug directly into the airways. Targeted delivery achieves higher concentrations at the site of action while minimizing the risks of systemic side effects.2,3 Data suggest that 50%–80% of patients are not using their inhaler correctly, possibly leading to suboptimal disease control, increased risk of exacerbations and hospitalization, and rising health care costs.3 Furthermore, therapeutic management of COPD often involves the use of multiple inhalers, and this increased complexity in treatment regimen decreases medication compliance, increasing the risk of poor disease management.3 In order to maximize adherence and treat COPD effectively, patient acceptance and ability to use their inhalers is a major factor.4 In developing new treatments for COPD, assessment of ease of use and patient preference for any new inhaler is important.5 Coformulation of treatments, to allow simultaneous delivery, reduces the number of inhalers required, simplifies treatment, and has been employed to address adherence issues.6 However, coformulation can be challenging with the potential for physicochemical interactions between drug entities.7 The ElliptaTM dry powder inhaler (DPI) contains two blister strips from which inhalation powders are delivered, enabling simultaneous delivery of two compounds without the need for coformulation. The ElliptaTM DPI inhaler design incorporated patient input in order to optimize patient handling and ease of use, with an overall aim of increasing patient compliance.8 This inhaler has been used in a series of clinical studies to deliver dual therapy of fluticasone furoate (FF)/vilanterol (VI) to patients with asthma (HZA106827; NCT01165138),9 and COPD;10–13 and umeclidinium bromide (UMEC) and VI dual therapy or component monotherapies to patients with COPD.14–18 High patient satisfaction was reported following FF/VI delivery via the ElliptaTM DPI in asthma and COPD, with both patient populations indicating a preference over their currently prescribed inhaler.19 This article describes the exploratory outcomes of the use of the ElliptaTM DPI during two 3-month double-blind, placebo-controlled Phase III trials of UMEC/VI, UMEC, VI, or placebo (all delivered by the ElliptaTM DPI). In these studies, correct usage and ease of use of the ElliptaTM DPI were assessed.20 Data from three 6-month double-dummy, active comparator trials (two Phase IIIa and one Phase IIIb trials) including UMEC/VI, UMEC, or VI (all delivered via the ElliptaTM DPI) and tiotropium (TIO; delivered by HandiHaler®) are also reported. In these studies, COPD patient preference for the ElliptaTM DPI or HandiHaler® was assessed.17,21 BODY.METHODS.STUDY DESIGNS: In all the five studies, treatment compliance with the ElliptaTM DPI was monitored using the inhaler dose counter. In the three active comparator studies, treatment compliance with the HandiHaler® was assessed by counting capsules returned. The study protocol and written informed consent form were reviewed and approved by the Chesapeake Institutional Review Board, as well as each relevant national, regional, or independent ethics committee or institutional review board, in accordance with Good Clinical Practice and the principles of the Declaration of Helsinki, with written informed consent obtained from all patients. BODY.METHODS.THREE-MONTH PLACEBO-CONTROLLED STUDIES: Two 3-month Phase III, placebo-controlled studies (DB2114417 [NCT01328444] and DB2114418 [NCT 01323660]) with a double-blind, incomplete block, two-period crossover design assessed once-daily (QD) UMEC/VI (125/25 mcg and 62.5/25 mcg), UMEC (125 mcg and 62.5 mcg), VI 25 mcg, and placebo; all delivered in patients with COPD, using the ElliptaTM DPI.20 Questionnaires assessing ease of use and ease of determination of the number of doses left in ElliptaTM DPI (Figure 1) were administered during the first study treatment period (at Day 43). Correct usage of the DPI was demonstrated/assessed through Visits 4–6 (at Days 1, 2, and 43) by staff at the study site. BODY.METHODS.ACTIVE COMPARATOR STUDIES: Inhaler preference was assessed using the COPD Device Preference Questionnaire (CDPQ; as described in the "Assessments" section) at Day 168 (Week 24) or the early withdrawal visit of two 6-month, Phase IIIa, blinded, double-dummy, parallel-group, active comparator studies (DB2113360 [NCT01316900] and DB2113374 [NCT01316913]) of UMEC/VI (125/25 and 62.5/25 mcg), UMEC 62.5 mcg, UMEC 125 mcg, and VI 25 mcg, administered QD, via the ElliptaTM DPI and TIO (18 mcg) administered QD via the HandiHaler®, in patients with COPD.17,21 During the studies, each patient used both inhalers daily, with one containing active treatment and the other placebo, depending on randomization. Since all the patients were using both inhalers throughout, it was possible to measure preference. The blinding process in these studies is described in the Supplementary materials; this did not affect the attributes tested by the CDPQ, although one question was removed as a result of the blinding process. The CDPQ was also used at Day 168 or early withdraw visit to assess inhaler preference in an additional 6-month, Phase IIIb, blinded, double-dummy, parallel-group, active comparator study (ZEP117115 [NCT01777334]) of UMEC/VI 62.5/25 mcg delivered QD via the ElliptaTM DPI and TIO 18 mcg delivered QD via the HandiHaler®.21 BODY.METHODS.ASSESSMENTS.ELLIPTA™ DPI USE AND EASE OF USE ASSESSMENTS (3-MONTH PLACEBO-CONTROLLED STUDIES): Correct DPI usage and ease of use were assessed in the first treatment period of these crossover studies. At randomization (Day 1) and Day 2, patients were trained in the correct use of the ElliptaTM DPI using the instructions provided in the patient information leaflet. Placebo inhalers were used for demonstration purposes in the training. Correct use of the ElliptaTM DPI involved three steps: 1) open the inhaler; 2) inhale the dose; and 3) close the inhaler. Following a demonstration of correct use by study staff, the patient's competence was assessed using a placebo inhaler. If the patient did not use the inhaler correctly, further instructions were given before assessing patient competence again. The inhaler demonstration was repeated up to three times on both the occasions until the patient could use the inhaler correctly. Patients who were not able to use the inhaler correctly after three demonstrations at randomization were ineligible to enter the study. Correct inhaler use was reassessed at Day 43 (after 6 weeks of treatment) using the demonstration inhaler, without further verbal instruction or demonstration. If the patient did not perform the maneuvers correctly at the 6-week visit, the procedure was demonstrated once again. At each assessment visit, the number of times the patient required additional instruction was recorded. The person providing training and assessing correct use of the inhaler was the same individual, where possible, for each patient. After 6 weeks of treatment, patients were asked to rate the ease of use of the inhaler by answering two questions: 1) How do you rate the ease of use of the inhaler? 2) How easily are you able to tell how many doses of medication are left in the inhaler? For each of the questions, answers were recorded using a 5-point difficulty scale: 1) very easy, 2) easy, 3) neutral, 4) difficult, and 5) very difficult. BODY.METHODS.ASSESSMENTS.CDPQ DEVELOPMENT: Prior to the active comparator studies, the CDPQ was developed and validated by RTI Health Solutions in collaboration with GlaxoSmithKline.22 Although it was not formally assessed or validated in any of the three active comparator phase III studies described, the CDPQ was used as a tool in these studies to assess inhaler preference among patients. A pool of potential questions for inclusion in the CDPQ was developed from market research with patients with COPD currently using inhaled therapy and with physician experts currently prescribing inhalers. Questions were based on the important characteristics of COPD inhalers identified by patients and physicians. Having recognized that "ease of use" was an important feature, a question format for the draft item set of the CDPQ was developed to assess inhaler preference based on this aspect. Questions relating to specific aspects of "ease of use" that could be affected by the overlabeling used in the blinding process were not included in the draft CDPQ. To refine and assess content validity, the draft CDPQ was then tested in 16 iterative cognitive interviews with adult patients with COPD.22 To assist understanding, patients were able to handle a placebo ElliptaTM DPI inhaler during the interviews, and all recruited patients were already being treated with, and hence were familiar with, the HandiHaler®. Participants in the first round of interviews (n=8) provided feedback on their preferred phrasing of the draft CDPQ. The CDPQ was then modified based on these responses, and a second round of interviews (n=8) was conducted during which participants assessed the modified CDPQ and provided additional input to confirm the content validity of the final version (shown in the Supplementary materials).22 The results of these interviews supported the content validity of the CDPQ, providing evidence that the measure adequately and appropriately assessed COPD DPI inhaler preference related to the most important ease of use concepts from the patient perspective. The second round of interviews also confirmed that the final wording of the instructions, the items, and their questionnaire responses were well understood by the patients with COPD providing evidence that the questionnaire adequately and appropriately assessed COPD DPI inhaler preference relating to ease of use.22 BODY.METHODS.ASSESSMENTS.INHALER PREFERENCE USING THE CDPQ (ACTIVE COMPARATOR STUDIES): The CDPQ was used on Day 168 (Week 24) or early withdrawal visit to assess inhaler preference in the three active comparator studies and consisted of three items that asked patients about their inhaler preference in terms of "number of steps," "time needed," and "ease of use." Choices of response included: preference for the ElliptaTM DPI, preference for HandiHaler®, or no preference. BODY.METHODS.STATISTICAL METHODS: The data presented here were prespecified as exploratory endpoints in each of the individual studies. For the purpose of this manuscript, the data were combined post hoc across the 3-month placebo-controlled studies and across the Phase IIIa active comparator studies, and all data were summarized descriptively. The Phase IIIb active comparator study was not integrated with the Phase IIIa studies because of being performed in a different timeframe and having a simplified study design compared with the Phase IIIa studies. No formal statistical analyses were performed. Unless otherwise stated, all summaries used the intent-to-treat population (all randomized patients that took ≥1 dose of study medication). BODY.RESULTS.PATIENT DEMOGRAPHICS: Patient demographics were similar between the two 3-month placebo-controlled studies;20 demographics were also comparable between the two Phase IIIa active comparator studies17 and between treatment groups in the Phase IIIb active comparator study.21 BODY.RESULTS.TREATMENT COMPLIANCE: Treatment compliance for the 3-month placebo-controlled studies and overall treatment compliance across the three active-comparator studies was high, with ≥84% patients being ≥95% (and ≤105%) compliant with study medication (Tables 1, 2, and S1). BODY.RESULTS.CORRECT USE, EASE OF USE, AND REMAINING DOSE DETERMINATION (3-MONTH PLACEBO-CONTROLLED STUDIES): Following the initial instruction on how to use the ElliptaTM DPI inhaler in each of the two 3-month placebo-controlled studies, of the 632 patients who took part in the study, 618 (98%) used the ElliptaTM DPI correctly at randomization on Day 1 (Table 3). In total (both studies combined), 14 (2%) patients required additional demonstration; of these, two patients required two additional demonstrations. Only one enrolled patient was excluded at randomization for failure of proper inhaler use (NCT01323660). When inhaler usage was reassessed at 6 weeks, 99% patients remaining in the study were using their ElliptaTM DPI correctly (Table 3). After 6 weeks, 99% of patients reported that they found the inhaler easy or very easy to use and 99% also reported that they found the dose counter easy or very easy to read (Table 3). Less than 1% of patients reported that they found the ElliptaTM DPI difficult to use, and no patients found the ElliptaTM DPI very difficult to use. BODY.RESULTS.INHALER PREFERENCE (ACTIVE COMPARATOR STUDIES): In the combined analyses from the two Phase IIIa active comparator studies, patients declared a preference for the ElliptaTM DPI compared with the HandiHaler® in relation to the number of steps to use (59% vs 17%), time taken to use (62% vs 14%) and ease of use (63% vs 15%), although 22%–24% of all patients declared no preference for either inhaler (Table 4). Results were similar in the Phase IIIb active comparator study (Table S2). These results were consistent regardless of which inhaler contained active drug in all three active comparator studies (Tables 4 and S2). BODY.DISCUSSION: This article describes the exploratory outcomes of ElliptaTM DPI usage, ease of use, and inhaler preference during five large Phase III trials of UMEC/VI in patients with COPD. In the 3-month placebo-controlled studies, following a simple training regime, the majority (98%) of patients demonstrated good technique using the ElliptaTM DPI after one attempt, with only 2% requiring further demonstration. After 6 weeks of use, 99% of patients were still using the ElliptaTM DPI correctly. Provision of a dose counter as a feature of inhaler design can help patients be aware when to seek a new inhaler, an important attribute for maintenance medication. In our studies, the majority (99%) of patients found the ElliptaTM DPI dose counter to be easy/very easy to read. Our results concur with a previous qualitative study conducted in patients with asthma and COPD, in which patients (n=75) reported high levels of satisfaction with the ElliptaTM DPI, describing it as straightforward to operate and easy to use.19 When interviewed, patients favored the size and mouthpiece fit over other inhalers.19 Dose counter visibility, simplicity of operation, and ease of use were also apparent drivers for patient preference over Diskus®, HandiHaler®, and various metered dose inhalers.19 In the three active comparator studies, patients expressed preference for the ElliptaTM DPI over the HandiHaler® regardless of whether the preferred inhaler contained active drug or placebo. These findings suggest independence from the efficacy outcomes reported in these studies, as the administration of placebo or three different active bronchodilator therapies via the ElliptaTM DPI did not impact the preference for this inhaler versus the HandiHaler®. Several other factors are known to influence treatment compliance in COPD, for example, suboptimal inhaler technique can result in poor disease control.4,23,24 Furthermore, if an inhaler is simple to use, patients are more likely to adhere to treatment regimens.2 Nonintentional noncompliance, that is, if a patient unknowingly makes mistakes during treatment administration, can be minimized by using easy-to-use inhalers and a less complex, easy-to-follow treatment regimen.2,24,25 The ability to provide single (UMEC) and dual therapies (FF/VI and UMEC/VI) using the ElliptaTM DPI supports this simplification. Intentional noncompliance with therapy, where a patient chooses to abstain from taking treatment, is less easily addressed by inhaler design alone although characteristics, such as shape, dimension, and cost may impact patient attitudes in taking treatment.2,25 Patients with COPD are a medically diverse group with variations in lung function, lifestyle, comorbidities, and intellectual ability impacting treatment choice and adherence.26 Patients with COPD tend to be older than their asthma counterparts and therefore more likely to have physical and cognitive impairments that may affect their ability to use an inhaler. Furthermore, these patients are more likely to have comorbid conditions and, therefore, take multiple medications,4,24,26 further highlighting the need for a simple treatment regimen with minimal number of inhalers. Other key factors relating to inhaler "misuse" include level of education and quality of instruction.23,24 Patient training often needs to be repeated in order to maintain correct inhaler technique.3 The results from the five assessed Phase III studies indicated that it was easy to learn and maintain the correct technique to deliver UMEC/VI using the ElliptaTM DPI. Although these studies included correct use of inhaler assessments and patient questionnaires, no direct measurement of inhaler technique was performed. Patient satisfaction with and preference for a specific inhaler can also impact adherence to therapy and consequently impact on long-term outcomes.4,5,25,27,28 Inhaler preference is now a recognized and valid patient-reported outcome for inclusion in clinical trials involving aerosolized medicines where a double-dummy design is used.25,27,28 However, a general lack of precision in measuring inhaler preference has been reported.28 Instruments, such as nonvalidated questionnaires and response scales, and questionnaires developed without input from relevant patients or psychometric testing have been commonly used,28 which may lead to increased variability in results. Validated questionnaires are now being developed, although the Patient Satisfaction and Preference Questionnaire is currently the only one with published validation.28 Measures of patient preference can only be obtained where study design provides patients' experience of the use of both inhalers requiring a double-dummy or crossover design. In these studies, the attributes of each inhaler to be compared need to be taken into account. Attributes that may be affected by any blinding process were not compared, to make the findings as generalizable as possible to routine clinical practice. Participant feedback on the recently developed CDPQ used in three of our studies suggests that the questionnaire items represent the most important concepts in determining COPD inhaler preference relating to ease of use concepts from the patient perspective.22 Furthermore, confirmation was obtained during its development that the final wording of the instructions, the items, and their responses were well understood by the patients with COPD, thus providing evidence that the questionnaire adequately and appropriately assessed DPI preference in patients with COPD relating to ease of use.22 These results were consistent across the three active comparator studies and show a preference for the ElliptaTM DPI over the HandiHaler®. The ElliptaTM DPI was not available in clinical practice at the time of the trials; hence, there was a possibility that patient familiarity with the HandiHaler® may have biased patient preference in favor of the HandiHaler®, although this was not assessed in these Phase III studies. Furthermore, the ElliptaTM DPI enables multiple dosage and does not require dose preparation, two factors known to have the greatest influence on patient preference.25 Treatment compliance was high in all five studies, and this is to be expected under the controlled environment of a clinical trial. However, compliance was assessed by dose counter or dose counting and no electronic registration of dosing was performed in these studies, which could be a limitation, and the usability of the ElliptaTM DPI should also be tested in the real-life setting of clinical practice. BODY.CONCLUSION: The majority of patients with COPD in these Phase III studies found the ElliptaTM DPI easy to use. In addition, the majority of patients expressed a preference for the ElliptaTM DPI over the HandiHaler®, potentially improving patient compliance and outcomes. Further assessment is warranted to determine whether this holds true in the clinical setting. BODY.SUPPLEMENTARY MATERIALS.METHODS.ACTIVE COMPARATOR STUDIES: In the three active comparator studies, a double-dummy design (described previously1,2) was used for dosing, whereby patients were given two inhalers, one containing active drug and the other placebo. All the patients and physicians were masked to assigned treatment during these studies; however, the tiotropium (TIO) capsules had trade markings that were not present on the placebo capsules, although they were closely matched in color.1,2 Both the TIO and placebo blister packages were covered with opaque overlabels in order to hide the information on the TIO packaging. The HandiHaler® dry powder inhalers were also covered with labels to mask the identifying marks on the inhaler. As the Phase IIIa studies were of parallel-group design, the capsule type was consistent for each patient for the duration of each of these studies.1 BODY.SUPPLEMENTARY MATERIALS.METHODS.COPD DEVICE PREFERENCE QUESTIONNAIRE DEVELOPMENT: The final version of the COPD Device Preference Questionnaire (CPDQ) is shown below: INSTRUCTIONS: Please complete the following questions related to both the Novel dry powder inhaler and Handihaler devices that you used during this study. Check only one response for each question. 1. Which device do you prefer based on the number of steps needed to take your COPD medication?□ No preference□ Handihaler device□ Novel dry powder inhaler device2. Which device do you prefer based on the time needed to take your COPD medication?□ No preference□ Handihaler device□ Novel dry powder inhaler device3. Which device do you prefer based on how easy the device is to use?□ No preference□ Handihaler device□ Novel dry powder inhaler device Table S1Summary of treatment compliance in the Phase IIIb active comparator study (117115; ITT population)Total N=905Compliancen883Compliance category, n (%)<808 (<1%)≥80 to <9545 (5%)≥95 to ≤105827 (94%)>105 to ≤1202 (<1%)>1201 (<1%)Notes: N is the number of patients in the ITT population; n is the number of patients with analyzable data.Abbreviation: ITT, intent-to-treat. Table S2Inhaler preference outcomes from using the COPD Device Preference Questionnaire in the Phase IIIb active comparator study (intent-to-treat population)Patients receiving active ElliptaTM N=454Patients receiving active HandiHaler® N=451Number of steps, n (%)n441434ElliptaTM262 (59)242 (56)HandiHaler®68 (15)68 (16)No preference111 (25)124 (29)Time needed to use, n (%)n441434ElliptaTM270 (61)256 (59)HandiHaler®65 (15)59 (14)No preference106 (24)119 (27)Ease of use, n (%)n441434ElliptaTM283 (64)256 (59)HandiHaler®61 (14)62 (14)No preference97 (22)116 (27)Notes: N is the number of patients in the ITT population; n is the number of patients with analyzable data.Abbreviations: COPD, chronic obstructive pulmonary disease; ITT, intent-to-treat. References1DecramerMAnzuetoAKerwinEEfficacy and safety of umeclidinium plus vilanterol versus tiotropium, vilanterol, or umeclidinium monotherapies over 24 weeks in patients with chronic obstructive pulmonary disease: results from two multicentre, blinded, randomised controlled trialsLancet Respir Med201426472486248358332Maleki-YazdiMRKaelinTRichardNZvarichMChurchAEfficacy and safety of umeclidinium/vilanterol 62.5/25 mcg and tiotropium 18 mcg in chronic obstructive pulmonary disease: results of a 24-week, randomized, controlled trialRespir Med2014108121752176025458157

TITLE: Clinical investigation of the acute effects of pomegranate juice on blood pressure and endothelial function in hypertensive individuals ABSTRACT.BACKGROUND: Pomegranate juice (PJ) is rich in bioactive phytochemicals with antioxidant, and anti-inflammatory and cardioprotective functions. The present trial investigated the acute effects of PJ consumption on blood pressure and markers of endothelial function. ABSTRACT.METHODS: In this single-arm study, thirteen hypertensive men aged 39-68 years were recruited. Included subjects were assigned to natural PJ (150 ml/day) following a 12 hour fast. Systolic blood pressure (SBP), diastolic blood pressure (DBP), and flow-mediated dilation (FMD), along with serum concentrations of C-reactive protein (CRP), intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), E-selectin and interleukin-6 (IL-6) were measured at baseline and 4-6 hours after PJ consumption. ABSTRACT.RESULTS: Comparison of pre- vs. post-trial values revealed a significant reduction in both SBP (7%; P = 0.013) and DBP (6%; P < 0.010). However, changes in FMD (20%) as well as circulating levels of CRP, ICAM-1, VCAM-1, E-selectin, and IL-6 did not reach statistical significance (P = 0.172). ABSTRACT.CONCLUSION: PJ has promising acute hypotensive properties. Consumption of PJ could be considered in the context of both dietary and pharmacological interventions for hypertension. BODY.INTRODUCTION: Cardiovascular disorders are among the leading causes of death and disability in the world.1,2 Hypertension is a major risk factor for cardiovascular and cerebrovascular disease, end stage renal disease, type 2 diabetes, and metabolic syndrome. Hypertension has a high global prevalence of about 15% that is estimated to reach as high as 30% by 2025.3,4 Controlled studies have indicated that each 5 mmHg decrease in diastolic blood pressure (DBP) is associated with 15% and 40% reductions in the risk of cardiovascular disease and stroke, respectively.5 In spite the introduction of several classes of anti-hypertensive agents with different mechanisms of action, uncontrolled hypertension resistant to drug therapy still remains a frequent medical problem. Pomegranate (Punica granatum L.; Family Punicaceae) is a popular edible fruit with wide applications in traditional medicine.6,7 Several lines of modern scientific evidence have also indicated the therapeutic efficacy of pomegranate against different types of disorders.8-11 The pomegranate is characterized by considerable amounts of biologically active phytochemicals including flavonoids (e.g. anthocyanins, catechins, quercetin, and rutin), other types of polyphenols, ellagitannins, and antioxidant vitamins.12-15 Many of these phytochemicals have been shown to possess antioxidant and anti-inflammatory properties plus additional biological activities such as inhibition of angiotensin converting enzyme.16-20 All these activities of the pomegranate are potentially beneficial for the treatment of hypertension and improvement of endothelial function.11 However, clinical studies investigating the hypotensive and cardioprotective effects of pomegranate have been scarce.20-26 According to the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC7), prehypertension is defined as 120 mmHg ≤ systolic blood pressure (SBP) < 140 mmHg 12 and/or 80 mmHg ≤ DBP < 90 mmHg.27 Prehypertension is a clinical stage where subjects are at increased risk (2 folds higher) of developing hypertension in the near future.28 Emerging findings suggest that interventions at the prehypertension stage can prevent or delay the progression of disease into established hypertension and subsequent detrimental outcomes.29 The present trial investigated the acute effects of pomegranate juice (PJ) on blood pressure and endothelial function in subjects with diagnosed prehypertension. BODY.MATERIALS AND METHODS: Subjects Thirteen hypertensive men aged 39-68 years were recruited for this trial.30 The Ethics Committee at the Shahrekord University of Medical Sciences (Iran) approved the study protocol (code: 92-3-16) and written informed consents were obtained from all participants. The inclusion criteria were body mass index (BMI) ≤ 30, and diagnosed hypertension defined as SBP > 120 mmHg and/or DBP > 80 mmHg. Exclusion criteria were type 1 or 2 diabetes, chronic pancreatitis, liver cirrhosis, kidney stones, renal failure, use of non-steroidal anti-inflammatory drugs, use of antioxidant or vitamin supplements, intense physical activity (> 5 h/week), smoking habit, being vegetarian or having any restrictive dietary requirements, and pregnancy. Study design The present study was designed as a single-arm clinical trial. The included subjects were assigned to natural PJ (150 ml/day) following a 12-h fast. SBP, DBP, and flow-mediated dilation (FMD), along with serum concentrations of C-reactive protein (CRP), intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin, and interleukin-6 (IL-6) were measured at baseline and 4-6 h after PJ consumption. Participants were asked not to eat or drink anything during the interval between PJ consumption and final measurements. Total anthocyanin assay A pH differential method was carried out to determine the total anthocyanin content of PJ. In this method, the difference in the absorbance of sample at pHs 1.0 and 4.5 is proportional to the total anthocyanin content. Details of this method have been published previously.24,31 Anthropometric and BP measurements Measurement of weight and height was carried out using a standard procedure as described previously.24,32 Body mass index (BMI) was calculated as weight in kg divided by height in meters squared (m2). BP was measured by a single operator at baseline and 4-6 h after intake of PJ, according to a standard protocol. BP recordings were performed after rest, employing a stethoscope and calibrated sphygmomanometer (Accurtorr 1A; Datascope, Japan). Systolic blood pressure was defined as the appearance of the first sound (Korotkoff phase 1) and diastolic blood pressure was defined as the disappearance of the sound (Korotkoff phase 5) during deflating of the cuff.24,32 FMD measurement Endothelium-dependent FMD was measured on the right brachial artery as described previously.33 All measurements were carried out by a single operator following a 5-min rest and employing a GE vivid 3 ultrasound apparatus (AtCor Medical, Solingen, Germany). A BP cuff was inflated around the forearm to 200 mmHg for 5 min. Images were recorded at baseline (before inflation), 30 s before cuff release, and then every 15 sec after cuff release for 3 min. Arterial diameter was measured at the end of end-diastolic phase, coinciding with R-wave on the electrocardiogram (ECG). Brachial FMD was expressed as the percentage change in arterial diameter from baseline.34 Blood sampling and biochemical analyses Twelve-hour fasted blood samples were taken from the left antecubital vein. After being allowed to clot for 2-3 h, serum was isolated by centrifugation at 3500-4000 rpm for 10 min. Serum samples were kept at -80°C prior to biochemical analyses. Biochemical analyses were performed using an automated enzymatic assay (Pars Azmoon, Tehran, Iran) on a Hitachi 902 autoanalyzer (for CRP), or enzyme-linked immunosorbent assay (ELISA) with commercial kits (Boster Biological Technology Ltd., Wuhan, China) (for ICAM-1, VCAM-1, E-selectin, and IL-6). Inter-assay coefficients of variation for ICAM-1, VCAM-1, E-selectin and IL-6 were 4.1-6.4%, 6.1-7.7%, 6.6-8.1%, and 3.1-5.5%, respectively. Intra-assay coefficients of variation for ICAM-1, VCAM-1, E-selectin, and IL-6 were 3.4-5.1%, 2.3-3.7%, 5.2-6.9%, and 2.3-4.9%, respectively. Statistical analysis All statistical analyses were performed using SPSS for Windows (version 17; SPSS Inc., Chicago, IL, USA). Data were expressed as mean ± SD. Group comparisons were made using paired t-test (in case of normally distributed data) or Wilcoxon signed-ranks test (in case of non-normally distributed data). A two-sided P-value of < 0.05 was considered to be statistically significant. BODY.RESULTS: This trial was a comprised of 13 hypertensive male adolescents with a mean age, weight, and BMI of 55.92 ± 7.92 yrs, 80.42 ± 11.01 kg, and 27.34 ± 3.82 kg/m2, respectively. All 13 subjects completed the study. Total anthocyanin content of PJ was determined to be 5.8 mg per 100 ml of the administered juice. Comparison of pre- vs. post-trial values revealed a significant reduction in both SBP (P = 0.013) and DBP (P = 0.010), amounting to an approximate reduction by 7% and 6%, respectively. However, percentage changes in FMD (20%) was not found to be statistically significant (P = 0.172). In the same manner, there was no significant difference in the circulating concentrations of inflammatory biomarkers namely hsCRP (P = 0.263), ICAM-1 (P = 0.248), VCAM-1 (P = 0.657), E-selectin (P = 0.182), and IL-6 (P = 0.763) following consumption of PJ. Baseline and post-trial values for the evaluated parameters are summarized in table 1. BODY.DISCUSSION: The present pilot trial is one of the few clinical evidences on the acute hypotensive and vascular effects of PJ. The results indicated amelioration of both SBP and DBP following consumption of a single dose of PJ. In a previous study, Aviram and Dornfeld investigated the effects of 2-week supplementation with PJ (50 ml/day) on the SBP of hypertensive patients.20 The findings revealed a significant decrease in SBP amounting to 5%. In the same study, a 36% decrement in the activity of serum angiotensin converting enzyme (ACE) was reported from PJ.20 The same group also investigated the effect of chronic supplementation with PJ (50 ml/day) in patients with carotid artery stenosis. Their results indicated a significant reduction in SBP, but not DBP, starting from 1 month after starting supplementation and generally increasing up to month 12 (equivalent to 12% decrement). However, no further reduction was observed when supplementation was continued for another 2 years.21 Another study conducted by Lynn et al. indicated a significant reduction in both SBP and DBP following consumption of PJ (330 ml/day) by healthy subjects for 4 weeks.22 Mathew et al. reported that consumption of pomegranate extract, either during or 15 min before a high-fat meal, can effectively prevent postprandial SBP rise at 2 and 4 h postprandial. Nevertheless, no significant effect was found on DBP.23 In a recent trial conducted by our group, the effects of a 2-week intake of PJ (from the same source as that used in the present study) were evaluated on BP of hypertensive subjects. Our results implied a significant reduction of both SBP (5%) and DBP (4.5%) compared to the control group who consumed water instead of PJ.24 Although most of the findings by previous trials infer the hypotensive impact of PJ consumption, there are some contrasting findings that are worth attention. Kelishadi et al. investigated the acute (4 h) and chronic (1 month) effects of PJ (240 ml/day) on BP of adolescents with metabolic syndrome and could not find any significant effect.25 In another trial, administration of PJ (240 ml/day) for 3 months was not found to significantly affect SBP or DBP in patients with stable coronary heart disease (CHD), as reported by Sumner et al.26 These negative findings might be attributed to the difference in the inclusion criteria applied by these two latter trials. It appears that hypotensive effects of PJ are more likely to be elicited in hypertensive patients, rather than patients with established CHD or metabolic syndrome. Moreover, the overall findings of the trials conducted so far weigh in favor of the beneficial effect of PJ consumption on BP. The hypotensive properties of PJ could be ascribed to the promising antioxidant properties of phytochemicals present in this complex juice. Oxidative stress is known to play a key role in the pathogenesis of hypertension.35 Increased levels of oxidants have been shown in different experimental models of hypertension.36-39 Detrimental effects of oxidative stress are mainly due to the interaction of reactive oxygen species (ROS) (in particular superoxide anion) with vital cellular components, lipids, and proteins, which leads to endothelial dysfunction and vascular resistance. In addition, ROS interfere with the production and vasodilatory actions of endothelium-derived nitric oxide (NO) via attenuating NO synthase (NOS) activity and enhancing NO breakdown.40,41 Epidemiological evidence has indicated that diets rich in natural antioxidants are associated with a reduced risk of developing hypertension and cardiovascular events.42,43 A plethora of studies have confirmed the considerable antioxidant and radical scavenging effects of PJ, which are mainly due to the anthocyanins and hydrolysable tannins present in the fruit. Interestingly, it has been suggested that some 50% of the total antioxidant activity of PJ is exerted by a specific ellagitannin, named punicalagin.16 Intestinal hydrolysis of punicalagin yields ellagic acid; the latter being a strong antioxidant compound.44 Apart from antioxidant properties, PJ may lower BP through a direct interaction with ACE. As referred above, a significant reduction in the activity of serum ACE has been observed in hypertensive patients following PJ consumption.20 Besides, in an animal study by Mohan et al. PJ administration attenuated angiotensin II-induced hypertension in diabetic rats, and also blocked the effects of different catecholamines on arterial BP and vasoreactivity. Furthermore, chronic administration of PJ counterbalanced the increased ACE activity in diabetic hypertensive rats.45 In spite of these positive reports, the ACE inhibitory effect of PJ needs to be further explored as Lynn et al. failed to report any change in serum ACE concentration following PJ consumption for 4 weeks.22 Another primary outcome measure that was evaluated by the present trial was changes in endothelium-dependent flow-mediated dilation. Our results did not indicate any improvement in FMD and this is in agreement with our recent report on the effects of 2-week PJ intake.24 However, findings from another trial indicated significant improvement in both endothelium-dependent and -independent (nitroglycerin-induced) dilation after 4 h of PJ consumption. In addition, this increased vasodilation was persisted until the end of supplementation period (1 month).25 In the present study, biomarkers of endothelial function, namely ICAM-1, VCAM-1, and E-selectin, remained statistically unaltered compared to baseline levels. It should be noticed that alterations in the circulating levels of these biomarkers is more likely to be exerted by chronic, rather than single dose, consumption of PJ and needs a longer term evaluation to allow the turnover of previously released proteins and observation of possible changes in the expression and subsequent release of these markers due to PJ consumption. This notion is corroborated by our previous study which showed a decreasing trend in serum levels of the aforementioned biomarkers following consumption of PJ.24 The present study has certain limitations that need to be acknowledged. This study did not include a control group. Therefore, our findings might have been confounded by bias. In addition, the present trial was conducted in pilot scale and with a small population size. This small size could potentially account for lack of detecting significant difference despite the increasing trend in FMD and decreasing trends in serum CRP, ICAM-1, VCAM-1, and E-selectin levels. With respect to these limitations, findings of the present trial may not be generalizable to the general population and should be interpreted with caution. In conclusion, the key finding to emerge from the present study is the acute hypotensive effect of PJ in hypertensive patients. While this trial is not a substitution for well-designed randomized controlled trials, it generates a hypothesis and motivates further research on this topic. Future large-scale investigations are indeed warranted in order to obtain a mechanistic understanding on this observed hypotensive activity.

TITLE: Push-out bond strength of intra-orifice barrier materials: Bulk-fill composite versus calcium silicate cement ABSTRACT: Background. The aim of this study was to compare the push-out bond strengths of calcium silicate-based ProRoot MTA and Biodentine cements and SureFil SDR and EverX Posterior bulk-fill composite resins. Methods. Twenty-four single-rooted maxillary central incisors were sectioned below the cementoenamel junction, and the root canals were instrumented using rotary files. Thereafter, a parallel post drill was used to obtain a standardized root canal dimension. The roots were randomly assigned to one of the following groups with respect to the intra-orifice barrier used: ProRoot MTA; Biodentine; SureFil SDR; EverX Posterior. Five 1-mm-thick sections were obtained from the coronal aspect of each root. Push-out bond strength testing was performed and data were analyzed with Kruskal-Wallis and post hoc Dunn tests (P<0.05). Results. SureFil SDR and EverX Posterior bulk-fill composite resins' bond strengths were significantly higher than ProRoot MTA and Biodentine calcium silicate cements. However, no statistically significant differences were observed between bulk-fill composite resins values and calcium silicate cement values. Conclusion. Within the limitations of present study, calcium silicate-based ProRoot MTA cement's push-out bond strength was lower than those of Biodentine, SureFil SDR and EverX Posterior materials. BODY.INTRODUCTION: Coronal leakage is one of the most important reasons for the failure after root canal treatment.1 Ray and Trope2 reported that the quality of coronal restoration is more important in protecting the periapical health than the quality of root canal filling. For this purpose, it has been suggested that to prevent the penetration of oral fluids and microorganisms into the root canals 3‒4 mm of coronal gutta-percha should be removed from the root canal and an intra-orifice barrier should be placed at canal orifice3 or a pulpal base should be placed using a restorative material.4 Previous studies have reported that covering the pulpal base with intra-orifice barrier materials after the root canal treatment constructs a secondary defense line against bacterial leakage.4,5 For this purpose, different materials have been employed, including temporary filling materials, glass-ionomer cement, composite resin, MTA and IRM.6,7 Today, MTA is also safely used in conservative pulpal treatments, root resorption treatments, and apexification procedures.8,9 MTA has been shown to be a bioactive material inducing the formation of hard tissues; it is well-tolerated by the tissues it contacts because of its biocompatibili.y10,11 However, long setting time of MTA (4‒6 hours) and the difficulties in adjusting the consistency while mixing make this material non-practical for clinical use.12 Numerous materials based on tri-calcium silicate have been developed and introduced to the market in order to eliminate these disadvantages of MTA.13-14 Biodentine (BD; Septodont, Saint-Maur-des-Fosses, France) is a new endodontic cement containing tri-calcium silicate and calcium carbonate, with a setting time of 12 minutes.15 The manufacturer claims that BD can be used as a replacement for dentin tissue for restorative purposes and as direct pulp cupping material for endodontic purposes, as well as restoration of perforations and as a root-end filling material.16 SureFil SDR flow (SDR; Dentsply Caulk, Milford, DE, USA), one of the bulk-fill composite resins recently introduced to the market, is a silorane-based nano- and micro-hybrid composite with low viscosity; its shrinkage stress is lower than conventional fluids.17,18 Another fiber-reinforced bulk-fill composite resin, also newly introduced to the market, is EverX Posterior (EXP; GC Dental Products Corp., Tokyo, Japan). In comprehensive literature research, no study was found, comparing the push-out bond strengths of SDR and EXP bulk-fill composite resins. For this reason, the aim of the present study was to compare the push-out bond strengths of calcium silicate-based ProRoot MTA and BD cements and SDR and EXP bulk-fill composite resins. The null hypothesis of the present study was there would be no statistically significant difference between the push-out bond strengths of the tested materials. BODY.METHODS. SPECIMEN SELECTION : Twenty-four single-rooted (0‒5°)19 maxillary central incisors, with no signs of calcification and extracted due to periodontal reasons, were included in the present study. The teeth were examined under ×2.5 magnification, and those with fractures or cracks or multiple apical foramina were excluded and replaced with new ones. In order to ensure the standardization, the crowns of the teeth were removed (by ensuring 16 mm of root length) with a fine diamond disc (Gebr. Brasseler GmbH & Co., Lemgo, Germany) at cementoenamel junction perpendicularly to the long axis of the teeth under water-cooling. BODY.METHODS. ROOT CANAL PREPARATION : Under ×2.5 magnification, the root canals of the teeth were penetrated using a #15 K-file (Dentsply Maillefer, Ballaigues, Switzerland); the file was inserted until it could be seen at the apical foramen, and then the working length was set at 1 mm shorter than this length. In order to shape the root canals, ProTaper Next (PTN; Dentsply Maillefer) rotary file system's X1, X2, X3 and X4 files were used respectively. The files were used at 300 rpm and 200 g cm-1 torque values in DR'S CHOICE program of VDW Reciproc Gold (VDW, Munich, Germany) endodontic motor. Each of the new file sets was used for shaping 4 canals and discarded. After each file, the root canals were irrigated with 2 mL of 5.25% NaOCl solution. Then, by using a parallel post drill with a diameter of 1.25 mm (ParaPost XT, Size 5; Coltene/Whaledent, Summit County, OH, USA), 10-mm-length gaps were prepared within the root canals. For final irrigation of the root canals, 2 mL of 17% EDTA (Vista Dental Products, USA) for 2 minutes and then 2 mL of 5% NaOCl for 2 minutes and 5 mL of distilled water were utilized. BODY.METHODS. PREPARATION OF THE SAMPLES FOR PUSH-OUT BOND STRENGTH TEST : Five 1-mm-thick transverse slices were taken under water-cooling (Isomet, Buehler, Lake Bluff, IL, USA) in corono-apical direction from each tooth. The slices were randomly divided into 4 groups (n=30). In the ProRoot MTA and BD groups, the materials were prepared in accordance with the instructions of manufacturer. The materials were placed on the dentin slices on a glass slab by using a hand plugger (Dentsply Maillefer), and the residual material was removed using a plastic spatula. In the SDR and EXP groups, the canals of the dentin slices were etched for 15 seconds using 35% phosphoric acid (3M ESPE, St. Paul, MN, USA), rinsed for 15 seconds and air-dried under low-level pressure of air (left in moist form). Two-step etch-and-rinse adhesive Prime & Bond NT (Dentsply DeTrey) was applied and kept for 20 seconds, and then the canals were dried with low-level pressure of air for 5 seconds and light-cured (Elipar S10; 3M ESPE) for 10 seconds. Dentin discs were filled using SureFil SDR and EverX Posterior on the glass slab, and then light-cured for 40 seconds (Elipar S10). All the prepared samples were kept at 37°C and 100% humidity for 7 days. The particulars of the tested materials are presented in Table 1. Table 1The Composition of the Tested Materials. Material Manufacturer Type Composition ProRoot MTA Dentsply, Tulsa Dental, USACalcium Silicate Cement Powder:Portland cement (75%), bismuth oxide (20%), calcium sulfate dihydrate (5%), tricalcium silicate, dicalcium silicate,tricalcium aluminate, tetracalciumaluminoferriteLiquid:distilled water Biodentine Septodent, Saint-Maur-des-Fosses, Cedex, FranceCalcium Silicate Cement Powder:tricalcium silicate, dicalcium silicate, calcium carbonate and oxide, iron oxide, and zirconium oxideLiquid:calcium chloride and hydrosoluble polymer SureFil SDR flow Dentsply, Tulsa Dental, USABulk-fill Composite Matrix composition:TEGDMA, EBADMAInorganic filler content:68 wt%, 44 vol%, barium borosilicate glass EverX Posterior GC EUROPE N.V., Leuven, BelgiumBulk-fill Composite Matrix composition:Bis-GMA, PMMA, TEGDMAInorganic filler content:74.2 wt%, 53.6 vol% Short E-glass berller, barium glass *PMMA, polymethylmethacrylate; bis-GMA, bisphenol-A-glycidyldimethacrylate; TEGDMA, triethylene glycol dimethacrylate; EBADMA, ethoxylatedbisphenol-A-dimethacrylate; wt%, weight percentage; vol%, volume percentage. BODY.METHODS. PUSH-OUT BOND STRENGTH TEST : After the samples set completely, each slice was fixed on a steel base with a hole in its center, and then connected to a universal test machine (Lloyd Instruments, Bognor Regis, England) (Figure 1). For push-out test, the stainless steel cylindrical tip with a 1-mm diameter was driven in apico-coronal direction at 1 mm/min crosshead speed until dislodgement. The Newton (N) values were converted into MPa values using the formula below: Bond strength (MPa) = Force for dislodgement (N) / Bonded surface area (mm2) Bonded surface area = 2×p×r×h (h: thickness of the dentin slice in mm; r: radius of the dentin slice canal in mm; p: constant: 3.14) Figure 1 The push-out testing device. BODY.METHODS. EVALUATION OF FAILURE PATTERNS : Following the push-out test, the slices were examined under a stereomicroscope at ×40 magnification to determine the nature of bond failure. Each sample was categorized into one of the three failure modes: adhesive failure at dentin‒material interface, cohesive failure within the material, or mixed failure, which is the combination of the two failure modes (Figure 2). The operator examining the slices was blinded to which sample matched which material. Figure 2 The images of the specimens before and after push-out test. (A) SDR group; (B) EverX Posterior group; (C) Biodentine group; (D) ProRoot MTA group; (E) Adhesive failure in SDR group; (F) Adhesive failure in EverX Posterior group; (G) Cohesive failure in Biodentine group; (H) Cohesive failure in ProRoot MTA group. BODY.METHODS. STATISTICAL ANALYSIS : The normality of data distribution was determined using Shapiro-Wilk test. Then the statistical differences between the groups were calculated using Kruskal-Wallis and post hoc Dunn tests. All the analyzes were performed using SPPS 21 (IBM-SPSS Inc., Chicago, IL, USA) software, and the statistical significance was set at 5%. BODY.RESULTS: The mean and standard deviation values of the tested materials obtained from the push-out bond strength test are presented in Table 2. SDR (4.10 ± 0.88) and EXP (3.86 ± 0.72) bulk-fill composite resins' bond strengths were significantly higher than those of ProRoot MTA (2.29 ± 0.43) and BD (3.20 ± 0.49) calcium silicate cements (P<0.05). However, there were no significant differences between bulk-fill composite resin values and calcium silicate cement values (P>0.05). Table 2Push-out bond strength values of tested intra-orifice barrier materials (MPa) Group Mean Standard Deviation ProRoot MTA 2.29 a 0.43Biodentine 3.20b 0.49SureFil SDR 4.10b 0.88 EverX Posterior 3.86 b 0.72 P-value < .05 a,b Different superscript letters indicate a significant difference between groups (P<0.05). The frequencies of fracture types in the test materials are presented in Table 3. No cohesive fracture was observed in SDR and EXP groups, while the mixed-type fracture was seen in the majority of cases. Mixed-type fracture was seen in 12 (40%) samples in the ProRoot MTA group and 15 (50%) samples in the BD group. Table 3Incidence of failure patterns of tested materials Adhesive Cohesive Mixed ProRoot MTA 16 (53.3%)2 (6.7%)12 (40%) Biodentine 14 (46.7%)1 (3.3%)15 (50%) SureFil SDR 8 (26.7%)0 (0%)22 (73.3%) EverX Posterior 11 (36.7%)0 (0%)19 (63.3%) BODY.DISCUSSION: The cements used for endodontic purposes must tightly bind to the root canal walls and resist tooth movements or mechanic stresses that may occur during treatment procedures.20-22 For this purpose, in the present study, the push-out bond strengths of ProRoot MTA, BD, SDR and EXP materials were compared. The bond of endodontic materials to the root dentin can be tested using different test methods such as traditional shear and push-out tests.23 Push-out test has been reported to be a reliable and practical test for examining the bond between materials and root dentin. In this test method, similar to the clinical environment, the fractures occur parallel to the resin‒dentin bond surface, and this method offers a better opportunity for analysis compared to the traditional shear test.24 For this reason, the push-out test was employed in the present study. The reason for obtaining 1-mm-thick dentin slices in the present study was that frictions that may cause misinterpretation of the results may occur in push-out test, and that it would be more reliable to utilize 1-mm-thick slices in order to minimize friction to eliminate this risk.23-25 According to the results of the present study, the push-out bond strength values of SDR, EXP and BD groups were significantly higher than those in the ProRoot MTA group. Therefore, the null hypothesis of the present study was rejected. Since there is no previous push-out bond strength study carried out using SDR and EXP bulk-fill composite resin in the literature, the results of the present study cannot be directly compared with other studies. However, the favorable outcomes of SDR might possibly be explained by its favorable stress behavior. In a recent study,17 a peculiar shrinkage behavior of the new flowable material in comparison with other flowable, nano- and micro-hybrid composites and a silorane-based material, was observed. The authors reported lower shrinkage stress, delayed point of gelation and lower shrinkage stress rates for SDR compared to the other materials that have been investigated. The high push-out bond strength of EXP material might be attributed to the ability of distribution of stresses occurring on the fibers distributed throughout the composite's matrix.26 Similar to the present results, it has been reported in many studies that BD has significantly higher push-out bond strength values than ProRoot MTA.27-30 Moreover, Silva et al31 and Centenaro et al32 reported that BD had significantly higher push-out bond strength than MTA Angelus (Angelus, Londrina, Brazil). In another study, Alsubait et al33 examined the push-out bond strengths of white MTA (WMTA; ProRoot, Dentsply Maillefer), BD and Bio Aggregate (BA; Innovative Bio Ceramix, Vancouver, Canada) cements and reported no significant difference between MTA and BD. The reason for BD's higher push-out bond strength compared to ProRoot MTA might be small molecular volume of BD cement and better penetration of cement into dentinal tubules, and consequently increased strength of bond to dentin. Moreover, because of this effect, the crystal formation might construct a dentin bridge within the dentinal tubules and thus the cement's mechanic retention might increase.16,34 According to the results of present study, the fracture modes of the samples generally were mixed. The good bond of materials to dentin, because of the high push-out bond strength values exhibited by BD, SDR and EXP groups, might explain this result. Similar to the results of the present study, Centenaro et al32 reported higher incidence of mixed-type fracture after they examined the fracture modes of MTA Angelus and BD cements after push-out test. However, the incidence of adhesive failure has been reported to be higher in other studies.27,35,36 These differences in the results might be explained by differences in methodologies used in sampling and preparation. BODY.CONCLUSIONS: Within the limitations of the present study, it might be concluded that calcium silicate-based ProRoot MTA cement's push-out bond strength was significantly lower than those of BD, SDR and EXP materials, with no differences between the push-out bond strength values of BD, SDR and EXP. BODY.ACKNOWLEDGMENTS: None. BODY.COMPETING INTERESTS: The authors declare no competing interests with regards to the authorship and/or publication of this article. BODY.ETHICS APPROVAL: The study protocol was approved by Ondokuz Mayis University ethics committee.

TITLE: The provision of written information and its effect on levels of pain and anxiety during electrodiagnostic studies: A randomised controlled trial ABSTRACT.OBJECTIVE: The provision of written information is a low-cost and readily available intervention that has been found to reduce pain and anxiety in a variety of clinical settings. The current study was undertaken to determine if information provision may improve patients' experience during conventional electrodiagnostic studies. ABSTRACT.METHODS: 128 participants were recruited from a tertiary teaching hospital who were referred for electrodiagnostic studies. They were randomized into 2 groups where the intervention group was provided with written information about the electrodiagnostic testing. Patients were invited to complete a questionnaire that included pain and anxiety using a visual analogue scale (VAS) following the testing. All participants underwent nerve conduction studies (NCS) whilst a subset also underwent subsequent needle electromyography (EMG). ABSTRACT.RESULTS: Those who received information had a statistically significant lower perception of anxiety during NCS, whilst only females who received information had a statistically significant lower perception of pain to both NCS and EMG. ABSTRACT.CONCLUSIONS: The provision of written information can reduce the degree of pain and anxiety experienced during electrodiagnostic testing. ABSTRACT.SIGNIFICANCE: Improving patient comfort and tolerability during electrodiagnostic testing may have practical implications towards more reliable and accurate results obtained from such investigations that may in turn improve patient diagnosis and management. BODY.INTRODUCTION: Due to the nature of electrophysiological evaluations, most patients express a degree of discomfort, pain and anxiety, with pain being the most common complication. This is well tolerated by most patients, however in some patients, poor tolerance may limit the validity and accuracy of the test by restricting the extent of the study and hence the diagnostic utility of these evaluations [1, 2]. This is particularly pertinent in the context of the ultimate value of these tests when taking into account the outcomes relative to the cost of the investigation. For a number of neuromuscular conditions, electrodiagnostic testing has been proven to have a dramatic impact on diagnosis and therapy [3]. Various interventions have been studied to increase tolerability of electrodiagnostic studies, particularly for pain related to needle electromyography (EMG). These include pharmacological interventions such as vapocoolant spray, eutectic mixture of local anaesthetics [4], ibuprofen [5] and paracetamol/tramadol [6]. Non-pharmacological interventions studied include listening to music [7], the provision of information [8], skin slapping [9], minimal insertion technique [10] to mention a few. The efficacy and safety of these have been well summarized in a recent article by London et al (2016) but the number of patients examined in these studies was small and none utilized a randomized study design. Providing information that includes both sensory (sensations that will be experienced) and procedural (the sequence of events) aspects of the procedure a patient will be undergoing has been found to be beneficial in reducing stress and pain [11–13] in a number of surgical settings. These findings have been shown in recent studies of pre-operative patients [14], hip replacement [15], oral surgery [16] and preoperative anesthesia [17] and postoperatively as part of a multimodal intervention for managing pain [18]. In the setting of electrodiagnostic testing, conflicting results have been found. One study [19] found that it had no important effect whilst an another older study found that it decreased pain perception during nerve conduction studies (NCS) but not EMG [8]. Furthermore, there have also been reports of differences in experience with electrophysiological testing amongst genders [20, 21]. Given this discrepancy, the current prospective study sought to investigate, using a randomized control design, whether written information given to a larger cohort of patients undergoing electrodiagnostic studies for the first time, is an effective means of reducing their subsequent perceived pain and anxiety levels. Differences in experience amongst male and female patients were also further explored. BODY.MATERIALS AND METHODS.PARTICIPANTS: This prospective study recruited patients whom were referred for outpatient NCS/EMG testing at a tertiary teaching hospital from October 2015 to December 2016. Participants were referred to the electrophysiology clinic for a wide range of provisional clinical diagnoses. These included most commonly entrapment neuropathies such as carpal tunnel syndrome, ulnar or peroneal neuropathy; peripheral neuropathy; radiculopathies; motor neuropathies or neuronopathies; and myopathy. Inclusion criteria were: age between 18–80 years inclusive, English literacy (sufficient for comprehension of information pamphlet and questionnaire), willingness to participate in the study, no active psychiatric illnesses or cognitive decline, and not taking anti-neuropathic agents or psychotropic drugs. Patients who met the above criteria were randomized on the day of testing by using a table of generated random numbers adapted from Pocock [22], where those assigned to even numbers received information whilst in the waiting room on the day of the study and those with odd numbers did not. Subjects were consented for the research study after NCS/EMG was completed. A total of 214 patients were initially recruited for the study over a one year period. Subsequent exclusions during data analysis were made based on the above-mentioned inclusion criteria or the questionnaire not being completed correctly. A total of 128 participants were included in the final data analysis and this was reached by further exclusion criteria (Fig 1). Amongst these, those participants who had previous NCS or EMG testing were excluded from the final cohort. In the final cohort, patients in the control group did not receive any self-reported additional information sources apart from basic verbal information provided by the examiner prior to the test. Patients in the intervention group would have received the information via the information brochure with or without further external information. 10.1371/journal.pone.0196917.g001Fig 1Flowchart of inclusion and exclusion criteria to achieve final cohort. BODY.MATERIALS AND METHODS.WRITTEN INFORMATION: Information was provided in the form of a 2 sided pamphlet informing patients about what to expect for the nerve conduction study, answering basic questions such as what the study would consist of, what patients needed to do during the test, what the test would feel like, how patients could prepare for the test, what to do if their symptoms were to get better before the test, how long the testing would take and whether it was safe if they had a pacemaker. On the second page, patients were informed that needle EMG may be performed subsequently and similarly, answered questions such as what it was, how many muscles would need to be tested, how they could prepare for it, what it would feel like, what they should expect after testing and when results would be received (S1 File). BODY.MATERIALS AND METHODS.BLINDING PROCEDURES, ELECTRODIAGNOSTIC EXAMINATION AND QUESTIONNAIRE: Electrodiagnostic examination was performed by skilled examiners guided by senior clinicians. Examiners who performed the study were blinded to the assignment of information. Examiners provided a standard introduction and brief explanation regarding the testing procedure to all patients prior to the commencement of the electrophysiological testing. In addition, all patients were also given the opportunity to ask questions regarding the testing procedure. All patients underwent NCS with or without subsequent EMG depending on the clinical indication. Electrical stimulation required for the NCS was standardised with the minimum stimulus required for an adequate examination as with typical studies. Immediately after the examination, patients who were eligible for the study completed a 2-page questionnaire in which they indicated their level of pain and anxiety experienced using separate 100 mm lines representing pain experienced during NCS, anxiety experienced during NCS, pain experienced during EMG and anxiety experienced during EMG, if applicable. The 100 mm line represented the visual analogue scale (VAS) scale (where 0 = no pain/anxiety on one end and 100 = worst pain/anxiety that they have ever experienced). The VAS scale, which is used as the primary outcome measure in this study, has been validated as a measure of the magnitude of experimental pain and anxiety [23–27]. The questionnaire included a secondary outcome measure of the experience of undergoing the study in relation to their expectation of the study. Further questions were whether they received information or had obtained information from other sources, whether they would have preferred to have received information, and the level of satisfaction with the electrodiagnostic examination. (S2 File). The study received approval from the New South Wales Health South Eastern Sydney Local Health District Human Research Ethics Committee and written consent was obtained from all participating individuals. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSES: Data analysis was performed using IBM SPSS 23 statistical package. The Student's t-test was used when the distribution of data in both intervention and non-intervention groups were normally distributed, while the Mann-Whitney U test was performed when the data groups were non-normally distributed. The data is expressed as mean ± Standard Error of the Mean (SEM) and the p-value cut off of less than 0.05 was considered statistically significant. The planned comparisons between the gender subgroups also utilized the Student's t-test or Mann-Whitney U test where appropriate. The Pearson chi-square test was used to study the differences in participant characteristics such as gender. It was also used to study the categorical data indicating patients' experience after undergoing the test in relation to their expectation in the intervention group compared to the non-intervention group. Correlation analyses were performed using Spearman rank test. BODY.RESULTS: Table 1 shows the baseline characteristics of participants in the NCS and EMG groups. There were no differences in age in the overall cohort (p = 0.13) as well as the EMG subgroup (p = 0.986), gender for the entire cohort (p = 0.22) and the EMG subgroup (p = 0.81), number of nerves studied (p = 0.88) and number of muscles (p = 0.08) sampled for both the intervention and non-intervention groups. 10.1371/journal.pone.0196917.t001 Table 1 Baseline characteristics of study population. Information received No information received p-value NCS N = 128 64 64 Age 53.34±2.05 49.22±1.75 p = 0.13 Gender (F/M) p = 0.22 Male 33 26 Female 31 38 Total NCS nerve studies 8.38±0.36 8.23±0.35 p = 0.88 Subgroup of patients who had subsequent EMG N = 28 15 13 Age 43.27±5.43 43.14±3.65 p = 0.99 Gender (F/M) p = 0.81 Male 11 9 Female 4 4 Total muscles sampled 4.40±0.76 2.77±0.38 p = 0.08 BODY.RESULTS.PRIMARY OUTCOMES: Overall scores for anxiety in the NCS cohort were found to be significantly lower (p = 0.04) in the intervention group (14.53±2.44mm) than in the non-intervention group (21.28±2.86mm; Fig 2). Further subgroup analysis did not find a significant information effect by gender although in females, there was a trend when comparing those who received information (14.68±3.64mm) to those that did not (25.15±3.93mm, p = 0.052). 10.1371/journal.pone.0196917.g002Fig 2There was a significant difference observed in the effect of information provision on the level of anxiety perceived during NCS compared to those that were not provided with information. Overall pain scores between the intervention (20.91±2.33mm) and non-intervention group (25.30±2.44mm) in the NCS cohort did not differ significantly (p = 0.15). Further gender subgroup analysis was performed but there were no significant differences in pain scores amongst males in the intervention group (22.56±3.03mm), compared to those in the non-intervention group (21.85±3.74mm; p = 0.71). However, amongst females, NCS pain scores were found to be significantly lower (p = 0.033) in the intervention group (19.15±3.60mm) compared to the non-intervention group (27.66±3.19mm; Fig 3). 10.1371/journal.pone.0196917.g003Fig 3There was a significant difference observed in the effect of information provision on pain perceived during NCS in females but not in males. No significant differences in the perceived pain (p = 0.34) or anxiety (p = 0.65) levels were demonstrated for the intervention and non-intervention groups in the subgroup of patients that underwent needle EMG testing. However, when further subgroup analysis by gender was performed, a significant information effect (p = 0.024) was found in females in the level of pain perceived during the EMG testing. Those who received information had significantly lower levels of pain (13.75±2.71mm) compared to those that did not (54.63±13.37mm) (Figs 4 and 5). 10.1371/journal.pone.0196917.g004Fig 4There was a significant difference observed in the effect of information provision on pain perceived during needle EMG testing in females but not in males. 10.1371/journal.pone.0196917.g005Fig 5There were no significant differences observed in the effect of information provision on the level of anxiety perceived during needle EMG testing. BODY.RESULTS.SECONDARY OUTCOME MEASURE: Amongst patients who received information compared to those who did not, those who received information showed a trend in reporting the study experience as being better than expected rather worse or same as expected (p = 0.055). Furthermore, females were significantly more likely to want the provision of information than men (p = 0.014). BODY.DISCUSSION: This is the first study utilizing a randomized control design in a large cohort of patients examining the effect of the provision of written information on the perceived levels of pain and anxiety during conventional electrodiagnostic testing. The use of VAS in retrospective pain assessments have been found to accurately reflect patients' pain experience during the procedure [28] and up to 3 months [29]. Concurrence in retrospective assessments in anxiety has also been similarly demonstrated [30]. The results of the current study demonstrated that compared with patients who were not provided with information, those who were provided with information showed a significant reduction in the anxiety levels perceived during NCS. It is interesting to note that for patients who received information compared to those who did not, there was a trend of reporting the study experience as being better than expected rather than worse or the same as expected. This is in keeping with previously reported data demonstrating that the amount of presented information allowed an increase in the perception of patient control that ultimately assisted in the management of acute procedural pain [31–33]. In females a reduction in pain levels was previously observed during NCS and EMG testing [20, 21]. While anxiety has been found to influence the level of pain experienced [34, 35], subgroup analyses assessing the effect of information on experienced levels of anxiety between genders did not reveal a statistically significant difference. This suggests that the reduction of pain experienced amongst females in our current study would not be adequately explained by a reduction in anxiety alone. Regarding information and cognitive control as it relates to the degree of experienced pain [36], it has been suggested that females have greater interest and concern regarding health matters [37]. As such, females in the current study who were not provided with prior written information, were significantly more likely to indicate that they would have preferred to have been given such information. Such an effect was not seen in males. The reduction in EMG pain perceived in females who received information compared to their male counterparts is consistent with the suggestion that there are gender differences in responses to non-pharmacological interventions such as cognitive intervention [38] for experimental pain [39]. Additionally, it has been suggested that predictors of effective treatment may be influenced by gender [39]. A much smaller sample of patients was collected for needle EMG compared to NCS and may represent a potential limitation of the study. The relative number of patients who went on to have EMG was small in our cohort as many of the referrals were for CTS, ulnar neuropathies, peripheral neuropathies, which in most clinical circumstances would not require needle EMG subsequent to the diagnostic NCS. Study group numbers for comparing pain in females during EMG was also small, however, it should be noted that means of groups were reflective of group perceptions. Future studies that are able to recruit larger numbers for needle EMG patients may be of benefit to corroborate the current findings. In conclusion, the provision of written information as a simple intervention to reduce subsequent pain and anxiety experienced during electrodiagnostic studies is effective, practical and easily implemented. Prior information is particularly effective in improving the tolerability of the study for females. This has important practical implications as improved tolerance to the testing may facilitate improved reliability and accuracy of electrodiagnostic results as well as allowing for more thorough testing which may in turn result in improved diagnosis and management for patients. BODY.SUPPORTING INFORMATION: S1 FileWritten information.Those participants whom were randomized to received written information were provided with this in print.(PDF)Click here for additional data file. S2 FileSample questionnaire.All participants whether provided with written information or now prior to the electrophysiological testing, were asked to complete the study questionnaire if they consented to take part in the study.(PDF)Click here for additional data file.

TITLE: Tramiprosate in mild-to-moderate Alzheimer’s disease – a randomized, double-blind, placebo-controlled, multi-centre study (the Alphase Study) ABSTRACT.INTRODUCTION: The aim of the study was to assess the clinical efficacy, safety, and disease-modification effects of tramiprosate (homotaurine, ALZHEMEDTM) in mild-to-moderate Alzheimer's disease (AD). ABSTRACT.MATERIAL AND METHODS: Double-blind, placebo-controlled, randomized trial in 67 clinical centres across North America. Patients aged ≥ 50 years, with mild-to-moderate AD (Mini-Mental State Examination score between 16 and 26) and on stable doses of cholinesterase inhibitors, alone or with memantine. Intervention: 78-week treatment with placebo, tramiprosate 100 mg or tramiprosate 150 mg BID. Measurements: Alzheimer Disease Assessment Scale – cognitive subscale (ADAS-cog) and Clinical Dementia Rating – Sum of Boxes (CDR-SB) assessments were performed at baseline and every 13 weeks. Baseline and 78-week magnetic resonance imaging (MRI) hippocampus volume (HV) measurements were conducted in a subgroup of patients. ABSTRACT.RESULTS: A total of 1,052 patients were enrolled and 790 (75.1%) completed the 78-week trial. Patient discontinuation and reasons for withdrawal were similar across groups. Planned analyses did not reveal statistically significant between-group differences. Lack of adequate statistical validity of the planned analysis models led to the development of revised predictive models. These adjusted models showed a trend toward a treatment effect for ADAS-cog (P = 0.098) and indicated significantly less HV loss for tramiprosate 100 mg (P = 0.035) and 150 mg (P = 0.009) compared to placebo. The incidence of adverse events was similar across treatment groups. ABSTRACT.CONCLUSIONS: The primary planned analyses did not show a significant treatment effect, but were confounded by unexplained variance. Post-hoc analyses showed a significant treatment-related reduction in HV loss. However, there was only a trend towards slowing of decline on the ADAS-cog and no slowing of decline on the CDR-SB. These results must be interpreted in consideration of the limitations of clinical and disease-modification outcome measures and their relationship, the heterogeneity of the disease and the impact of confounding demographic and clinical variables. BODY.INTRODUCTION: Current therapies for Alzheimer's disease (AD) are symptomatic with limited impact on the disease itself. Treatment that slows or stops disease progression remains an unmet need. Accumulation of the amyloid β (Aβ) peptide is a pivotal process in the natural history of AD. Amyloid β is generated by enzymatic cleavage of the amyloid precursor protein (APP) with subsequent formation of toxic Aβ oligomers and amyloid fibrils, which are ultimately deposited as plaques in the brain [1]. This process is hypothesized to induce neurotoxic events that lead to neurovascular uncoupling, synaptic dysfunction and neuronal loss [2, 3]. Ensuing structural damage in some brain areas such as the hippocampus, as revealed by magnetic resonance imaging, may be associated with, or even precede, clinical symptoms [3]. Interventions that protect against Aβ-induced neurotoxicity may have therapeutic value for the treatment of AD [4]. Tramiprosate (homotaurine, ALZHEMEDTM) is a small, orally-administered compound that binds to soluble Aβ and reduces amyloid aggregation and subsequent deposition [5]. In vitro, tramiprosate provides neuroprotection against Aβ-induced neurotoxicity in neuronal and mouse organotypic hippocampal cultures, and reverses Aβ-induced long-term potentiation (LTP) inhibition in rat hippocampus [6], in part, through activation of β-aminobutyric acid A (GABA-A) receptors [7]. In vivo, tramiprosate produced dose-dependent reductions of Aβ in the brain of transgenic mice (hAPP-TgCRND8) [6]. Clinical studies [8] showed that tramiprosate was safe and tolerable. In mild-to-moderate AD patients [8] tramiprosate also reduced Aβ42 levels in CSF. The objectives of the Alphase, Phase III, North-American multi-centre, randomized, double-blind, placebo-controlled study was to assess the clinical efficacy, safety, and disease-modification effects of tramiprosate in patients with mild-to-moderate AD. BODY.MATERIAL AND METHODS.PATIENTS: Participants (men and women ≥ 50 years) had a diagnosis of probable AD (DSM-IV-TR [9] and NINCDS-ADRDA [10]) and a Mini-Mental State Examination (MMSE) [11] score between 16 and 26. Laboratory assessments, electrocardiograms (ECG) and CT/MRI results were compatible with probable AD. Patients were required to be on a stable dose of a cholinesterase inhibitor (ChEI) which may have been combined with memantine, for a minimum of four months prior to the screening visit. Stable doses (≥ 1 month prior to screening) of anxiolytics, sedatives, hypnotics, antidepressants, antipsychotics, anticonvulsants, oestrogens, statins, and vitamin E (≤ 2050 IU/day) were allowed. Patients with any other causes of dementia were excluded, as were those with a body mass index < 19 or > 28, a life expectancy < 2 years, or a clinically significant and uncontrolled medical disease. The study protocol was approved by the ethics review board of each site. Written informed consent was obtained from each patient or legally authorized representative prior to study entry. BODY.MATERIAL AND METHODS.STUDY DESIGN: This was a randomized, double-blind, placebo-controlled, parallel-group study conducted at 67 study centres across the United States and Canada. Patients were randomized to tramiprosate 100 mg BID, tramiprosate 150 mg BID, or placebo BID for 78 consecutive weeks, including an 8-week dose escalation phase. Treatment was allocated according to a randomization list issued by an independent biostatistician, using a computer random number generator, and balanced to ensure a ratio of 1 : 1 : 1 across groups. Study medication consisted of modified-release coated tablets, identical in external appearance and containing 50 mg of tramiprosate or placebo. All doses were administered orally as three tablets. BODY.MATERIAL AND METHODS.PROCEDURE: Clinical efficacy measures were Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog) [12], Clinical Dementia Rating-Sum of Boxes (CDR-SB) [13], MMSE [11], Clinical Interview Based Impression of Change-plus caregiver interview (CIBIC-plus) [14], Neuropsychiatric Inventory (NPI) [15], and Disability Assessment of Dementia (DAD) [16] scores. Disease-modification measures were volumetric magnetic resonance imaging (vMRI) of the hippocampus, whole brain and entorhinal cortex, plasma/CSF/urine Aβ, and CSF tau. Safety measures included Adverse Event (AE) reporting (MedDRA 9.0), vital signs, clinical laboratory parameters, ECG, and physical examinations. Clinical and safety assessments were conducted at Baseline and Weeks 13, 26, 39, 52, 65 and 78. HV was measured at Baseline and at Week 78 in a subgroup of patients at 51 sites. The complete description of HV measurements is provided elsewhere [17]. Briefly, centralized assessment of HV was derived from a 1.5T T1-weighted 3-dimensional volumetric MR sequence (TR = 10/2160/9.5 ms, TE = 6.8/3.9/4 ms, 192 × 256/192 × 256/256 × 256 matrix, 260/260/258 mm FOV, flip angle = 20°/10°/8°, slice thickness = 1.6/1.6/1.6 mm for GE/Siemens/Philips, respectively). HV was computed using a semi-automated atlas-based warping method [18]. BODY.MATERIAL AND METHODS.STATISTICAL ANALYSES: In this report, only the ADAS-cog, CDR-SB, and HV results will be reported as they constituted the primary clinical efficacy and disease-modification endpoints. Primary clinical efficacy endpoints were the changes from Baseline to Week 78 in ADAS-cog and CDR-SB scores. Secondary efficacy endpoints were the changes from Baseline to Weeks 13, 26, 39, and 52 in ADAS-cog and CDR-SB. Primary disease-modification endpoints were the changes from Baseline to Week 78 in vMRI of the hippocampus. Sample size calculations were based on the assumptions that after 78 weeks, patients on placebo would decline by a mean (SD) of 10 (8.0) points on the ADAS-cog and 3.0 (2.3) points on the CDR-SB [19]. Assuming a 25% effect size, the study was powered to detect a difference of 2.5 points on the ADAS-cog and 0.75 point on the CDR-SB with 90% power at a two-tailed 5% significance. Adjusting for an approximate 30% dropout rate, an enrolment of 315 patients per group was planned. For HV, sample size estimate was based on an untreated mean (SD) 5.2% (2.5%) annual change in HV [20-25]. In order to detect a 20% difference in the HV change between active and placebo with 80% power, at a 5% two-tailed significance, and assuming an approximate 30% patient discontinuation rate, 150 patients per group were to be enrolled in this sub-study. BODY.MATERIAL AND METHODS.PLANNED MODELS: Changes from Baseline in ADAS-cog and CDR-SB scores were assessed using mixed-effect repeated-measures analyses of covariance (ANCOVA). These models tested for the main effects of Group (treatment arm), Visit (treatment duration) and Group × Visit (G × V) interaction, adjusted for Baseline values and Site as covariates. The G × V interaction reflected between-group differences in least-square (LS) mean changes over time. Between-group differences were determined using planned contrasts comparing each active treatment group to placebo with respect to the changes in ADAS-cog and CDR-SB from Baseline to Week 78 (primary clinical outcome) and from Baseline to Weeks 13, 26, 39, 52, and 65 (secondary clinical outcomes). Change in HV was calculated by subtracting the final visit volume from the Baseline volume using the Last Observation Carried Forward approach for withdrawn patients. Differences in changes over time in HV between treatment arms were evaluated with an ANCOVA in which the dependent variable was the change in HV over the 78 Weeks of the study and the independent variables were treatment group with three levels (tramiprosate 150 mg BID, tramiprosate 100 mg BID, and Placebo BID), duration of follow-up (in months), Baseline HV and Site. Between-group differences in the change in HV were assessed with the overall F-test for treatment group while differences between each active group and placebo were assessed using planned contrasts. BODY.MATERIAL AND METHODS.ADJUSTED MODELS: For all study outcomes, unanticipated and highly significant Site effects (P < 0.001) were detected in the planned ANCOVA models. This suggested that either a significant amount of the variability was due to differences between sites, or that "Site" was concealing confounding variables that were not accounted for in the model, contributing to poor model fit. Post-hoc analyses aimed at reducing Site effects and improving model fits were conducted. Adjusted mixed-effects multivariate models were developed using aggregate data. Additional covariates were selected using a four-step process: 1) identification of cross-sectional and longitudinal patterns in the data, 2) identification of variables which were predictors of outcome based on a statistical trend defined as a P < 0.15, 3) selection of variables that were significant independent predictors of outcome, with a P < 0.15 tolerance, and 4) assessment of the clinical relevance of the identified variables by an independent scientific advisory committee. The fixed effect covariates in the planned models were removed based on likelihood ratio test and information criteria improvement (Akaike and Bayesian). Once the final adjusted models were developed, treatment variables were included in a three-level linear mixed-effect model with random site-specific intercepts and slopes for ADAS-cog and CDR-SB, and a two-level linear mixed model with random site-specific intercepts for HV. In the adjusted models, contrasts comparing each active group to Placebo at each visit were exploratory and aimed at documenting possible trends. No adjustments for multiple comparisons were thus required. Statistical trends (P < 0.15) are reported. Both planned and adjusted models were based on observed cases. Efficacy analyses were conducted in the Intent-to-Treat (ITT) population, defined as all treated patients who had a Baseline ADAS-cog or CDR-SB assessment and at least one corresponding post-Baseline assessment. Disease-modification analyses were based on the MRI-ITT population, defined as ITT patients with a Baseline and on-treatment MRI that was valid for evaluating at least one of the targeted brain structures. Sites that recruited fewer than six patients were pooled. Twelve patients were excluded due to GCP non-compliance. Statistical analyses were performed using SAS version 9.12. BODY.RESULTS.PATIENT DISPOSITION AND BASELINE CHARACTERISTICS.: The study took place between August 2004 and February 2007. A total of 1,432 patients were screened, of whom 1,052 were randomized (placebo BID: n = 353, tramiprosate 100 mg BID: n= 352, tramiprosate 150 mg BID: n= 347) and 790 (75.1%) completed the trial. Patient discontinuation and reasons for withdrawal were similar across groups. Adverse event (48.1%) and withdrawal of consent (30.5%) were the most frequent reasons for early discontinuation. Patient disposition is presented in Figure 1. There were no statistical differences between groups with regards to baseline and demographic characteristics (Table I). BODY.RESULTS.PLANNED ANALYSES: Table II summarizes results using the planned analyses. All three groups experienced an increase from Baseline to each visit in clinical scores, indicating deterioration. The planned ANCOVA model for ADAS-cog did not reveal a significant G × V interaction (P = 0.152) although planned contrast for the primary ADAS-cog outcome revealed numerically less decline at Week 78 in the 100 mg versus placebo (–0.70 points). Neither the planned G × V interaction (P = 0.573) nor the planned contrast for the primary CDR-SB outcome at Week 78 (100 mg vs. Placebo: P = 0.405; 150 mg vs. Placebo: P = 0.837) revealed a statistically significant treatment effect. Planned contrasts for the secondary ADAS-cog outcomes revealed that, relative to placebo, the 100 mg BID group had a statistically lower change from Baseline (P = 0.027) at Week 26. No statistical difference relative to placebo was observed for any of the secondary CDR-SB outcomes. For HV change, the planned model showed a non-significant treatment effect (P = 0.120). Compared to Placebo, the 100 mg BID group showed no volume change difference (P = 0.782), while a trend toward greater HV decrease was observed in the 150 mg BID group (P = 0.056). BODY.RESULTS.ADJUSTED MODELS: The adjusted models (Table III) improved data fit as indicated by Aikaike Information Criteria (AIC) values of 28772.5 for the ADAS-cog and 17355.4 for the CDR-SB, compared to 31559.2 and 20030.3, respectively, for the planned models. The ADAS-cog adjusted model revealed a statistical trend for treatment effect in favour of the two tramiprosate groups (G × V interaction: P = 0.098) while post-hoc planned contrasts showed lower changes from Baseline relative to placebo in the 100 mg BID group at Week 26 (P = 0.065) and Week 52 (P = 0.090). The adjusted model for CDR-SB revealed no significant treatment effect (G × V interaction: P = 0.505). For HV, the adjusted model showed an improved AIC of 3600.3 compared to 8139.1 for the planned model. The following covariates and covariate interactions were retained: Antidepressant Use, Vitamin E Use, Genotype, Cardiovascular Disease, Race, Type of ChEI Used, Total Intracranial Volume, Whole Brain Volume at Baseline, Baseline Hippocampus Volume × Treatment, Age Group × Treatment, Genotype × Treatment, Race × Treatment, Month × Genotype, Month × Cardiovascular Disease, Month × Race, and Month × Total Intracranial Volume. The HV adjusted model showed a significant treatment effect (P = 0.011). Least square estimates of the mean [95% CI] changes from Baseline were significantly different from zero for the placebo (–419.3 mm3 [–642.1 to –196.4 mm3]; P < 0.001) and 100 mg BID (–135.1 mm3 [–249.5 to –20.7 mm3]; P = 0.021) groups, while the change of 79.5 mm3 (–182.5 to 341.5 mm3) observed for the 150 mg BID group was not (P = 0.550). The decrease in HV were significantly less than placebo for both the 100 mg (P = 0.035) and 150 mg (P = 0.009) BID groups. BODY.RESULTS.SAFETY: Table IV summarizes the most common adverse events (AE) and serious adverse events (SAE) of all causalities. The proportion of patients experiencing at least one AE for the placebo, 100 mg BID and 150 mg BID groups was 92.1%, 95.2% and 94.8%, respectively. Nausea, syncope, vomiting, and weight decrease appeared to be dose-related. However, the incidence of these AEs in the 100 mg BID group was not statistically different from placebo. A total of 255 patients (24.2%) experienced at least one SAE with an incidence of 26.1%, 23.6% and 23.1%, for the placebo, 100 mg and 150 mg BID groups, respectively. Syncope and pneumonia were among SAEs that appeared to be dose-related. Thirty-two (3.0%) patients experienced an AE which resulted in death. Mortality rate was higher in the placebo group (4.0%) compared to the 100 mg (2.8%) and 150 mg (2.3%) BID groups. Gastrointestinal disorders were among the most frequent AEs that led to discontinuation, with a total of 38 (3.6%) patients. When examining the AE profile among the most frequently occurring AEs that led to discontinuation, nausea (Placebo: 1 [0.3%]; 100 mg BID: 3 [0.9%]; 150 mg BID: 10 [2.9%]) and vomiting (Placebo: 0 [0%]; 100 mg BID: 4 [1.1%]; 150 mg BID: 7 [2.2%]) appeared to be dose-dependent events. Laboratory test results were generally unremarkable, with no apparent clinical differences between groups. BODY.DISCUSSION: In this trial of tramiprosate in mild-to-moderate AD, the planned analyses did not show statistically significant between-group differences. Significant Site effects and the impact of variables not included in the planned analyses reduced model fit and validity. In addition, reduced power due to within- and between-patient variance, as well as the unexpected low deterioration in the placebo group, render these analyses inconclusive. Results of the adjusted models, which improved statistical fit and reduced the impact of Site effects, revealed a statistical trend toward a treatment effect on cognition and a treatment-related reduction in HV loss. Overall, tramiprosate was well tolerated. AD typically progresses from mild functional impairment to total dependence and death over a period of 5-20 years. A disease-modifying treatment initiated in the mild stage that slows progression by 25% would be expected to have a significant clinical impact. Consistent with this goal, and based on results from earlier trials, our study was powered to demonstrate a 2.5 point treatment effect on the ADAS-cog at 18 months. Although we had estimated an approximate 10-point deterioration in the placebo group, the 18-month non-adjusted and adjusted changes in ADAS-cog were only 7.4 points and 6.2 points, respectively. Accordingly, a 25% difference from placebo would be equivalent to an unadjusted change of 1.8 points and an adjusted change of 1.6 points. This trial was thus underpowered to detect a treatment effect, an issue of importance to other disease-modifying drug development programmes. Statistical powering may also be affected by variance in longitudinal outcome measures that could obscure potential treatment signals and bias results toward the null hypothesis. In AD, many factors, such as concomitant medication type and dose, can affect outcome variability and their combined impact likely increases as trials become longer and involve larger cohorts. Simple ANCOVA models, such as those used in the planned analyses, may not have been sufficient to model unexplained variance arising from uncontrolled conditions. Indeed, the high unexplained variance found in the planned statistical models was the impetus for the development of more complex mixed-effects models that included multiple covariates and yielded improvement in modelling fit. This underscores the complexity of AD and the importance of careful consideration of statistical modelling aimed at improving statistical fit and validity of results. Results of the HV analysis illustrate the importance of statistical modelling. Our planned analysis showed a trend toward greater HV loss in the 150 mg BID tramiprosate group, a finding consistent with the AN-1792 amyloid vaccine trial, which showed a non-significantly greater HV loss in the antibody responders [26]. While the underlying neurological basis of the HV loss in the vaccine trial remains unclear, inclusion of covariates selected on the basis of clinical relevance and statistical significance in our trial reversed the direction of the between-group difference, with no change in HV in the 150 mg BID group and a lower volume change relative to placebo in the 100 mg BID group. Interestingly, the adjusted model identified a significant interaction between treatment and several covariates, specifically, age, hippocampus volume at baseline and APOE genotype. These variables have previously been found to exert an impact on HV in a number of longitudinal imaging studies [27-29]. Our study suggests that these variables may modulate the effects of an anti-amyloid intervention on HV changes over the course of the trial. Confirmation of the impact of the above covariates and their interaction with treatment in other data sets would corroborate our findings and allow the development of more complex imaging analysis plans in future trials. In our study, we did not find close agreement between cognitive/clinical and HV changes. Psychometric tests and vMRI may not measure precisely the same disease processes, and changes in biological and cognitive measures may not occur over the same time period [17, 30]. Nevertheless, volumetric MRI and cognitive measures are complementary and may be implemented in AD trials in order to document potential treatment effects on the underlying disease and to provide regulatory support for disease modification claims. This trial is among the first published Phase III trials of anti-amyloid compounds for the treatment of AD. Planned analyses did not demonstrate statistically significant between-group differences, but were confounded by weakness of the statistical models. Post-hoc adjusted analyses showed a treatment effect in reducing HV loss and a trend toward reduced cognitive decline. These results must be interpreted in consideration of the limitations of the outcome measures, the heterogeneity of the disease and the impact of demographic, genetic and clinical variables on patient outcome.

TITLE: The feasibility and acceptability of neuromuscular electrical stimulation to improve exercise performance in patients with advanced cancer: a pilot study ABSTRACT.BACKGROUND: To determine the feasibility and acceptability of lower limb neuromuscular electrical stimulation (NMES) as a home-based exercise therapy in patients with cancer who could not attend hospital-based exercise training. ABSTRACT.METHODS: A single-arm prospective pilot study of NMES, applied daily to both quadriceps muscles for six weeks. Participants were recruited from patients referred to a hospital-based multi-disciplinary supportive care team specializing in treatment of patients with nutritional depletion and functional decline. ABSTRACT.RESULTS: Of the 15 participants who underwent baseline testing, 10 (67%) completed the study and only one (7%) withdrew because of discomfort due to NMES treatment. 7/10 (70%) of participants used NMES at least three times a week for the duration of the study. Use of NMES did not lead to significant improvements in physical performance tests. ABSTRACT.CONCLUSIONS: NMES is a feasible and acceptable intervention for home use in patients with cancer, poor performance status and metastatic disease. However, whether NMES is an effective strategy to stabilize or improve physical performance in such patients is not proven. BODY.BACKGROUND: Patients with cancer frequently suffer problems common to other chronic diseases, namely long-lasting sequelae from their disease and its treatments [1] as well as progressive decline in overall functional status [2,3]. In many chronic medical conditions e.g. chronic obstructive pulmonary disease (COPD), and congestive heart failure (CHF), exercise-based rehabilitation programs have a major impact on quality of life and functional status [4] and are widely deployed as part of standard clinical care. However, despite growing evidence that exercise training in cancer patients also contributes to improved quality of life and tolerance of anti-cancer treatment [5], exercise-rehabilitation has not been generally adopted for patients with cancer. Exercise training programs for patients with chronic diseases typically involve hospital-based supervised training two or three times a week. However, attendance at hospital-based exercise training is difficult for debilitated patients with cancer; and though patients with advanced cancer are willing to exercise they strongly prefer to do so at home [6,7]. However the beneficial impact of such home-based training is still unclear and is difficult to measure. For example, a recent study of home-based semi-supervised exercise was unsuccessful because of unexpected poor accrual and high dropout rates [8]. Outside of such studies, community or home-based supervised training is often not readily available for patients with medical conditions. Thus by default, a self-directed unsupervised home exercise program is often recommended for patients unable or unwilling to attend hospital training, despite the inherent difficulties in monitoring exercise adherence and intensity. Other approaches are therefore needed which can deliver a safe and effective exercise stimulus at home, in a manner which is suitable for use by patients with poor physical function. Neuromuscular electrical stimulation (NMES) delivers a controlled contractile stimulus to underlying muscles via surface electrodes placed on the skin. It is widely used for rehabilitation of neurological or orthopedic conditions. NMES is often used to assist with rehabilitation following joint surgery [9], by helping to speed up the recovery of muscle strength and joint function. Moreover, NMES used in conjunction with voluntary contraction exercises, accelerates leg muscle strengthening after knee arthroplasty [10]. More recently NMES has been assessed for patients suffering chronic medical conditions. In debilitated patients with COPD or CHF, NMES of the large lower limb muscles was shown to increase muscle size [11], improve muscular strength and overall exercise capacity [12-14], facilitate activities of daily living, and improve quality of life [15]. Despite the evidence for effectiveness of NMES in other patient populations, relatively few studies have been performed to test the potential usefulness of NMES in patients with cancer. One case study reported use of NMES in a single patient with extensive metastatic lung cancer, who achieved improvements in mobility, function and quality of life with NMES [16]. Another pilot study used NMES in a randomized trial involving 16 advanced stage non-small cell lung cancer patients with good performance status. Those receiving NMES found it highly acceptable, but there was only a non-significant trend towards improvement in leg muscle strength or other measures of endurance and habitual daily activity [17]. In a follow-up phase II study, the same authors recruited lung cancer patients undergoing first-line palliative chemotherapy and randomized them to NMES intervention (30 patients) or a control (19 patients) group [18]. In this later study there was no improvement in the physical function tests (quadriceps strength or physical activity), and a lower than expected level of adherence. As a result, the authors concluded that NMES does appear to warrant further study in advanced lung cancer patients at this stage in their treatment [18]. This does not rule out a role for NMES to reduce muscle loss and dysfunction [19] in other patients with cancer, but to date there is still little published data on the use of NMES in patients with cancer in general, especially those with poor performance status. The McGill Cancer Nutrition-Rehabilitation Program clinic at the Jewish General Hospital (CNR-JGH clinic) in Montréal, is a multi-disciplinary team that specifically addresses the clinical needs of patients with cancer referred with weight loss, anorexia, and reduced physical function. The CNR-JGH clinic uses a comprehensive interdisciplinary approach to assess and control symptoms, along with individualized dietary interventions and exercise training wherever possible. In our experience, hospital-based exercise training is very difficult to deliver to patients with poor performance status and those who live far away from the hospital. To address this practical problem, a pilot study was performed to evaluate the acceptability and feasibility of a home-based NMES intervention in a heterogeneous group of patients attending the CNR-JGH clinic, the majority of whom had advanced stage cancer. The secondary objective of the study was to assess the impact of the NMES intervention on tests of physical function focused on walking endurance capacity, lower extremity strength and global functional performance. BODY.METHODS.PATIENT RECRUITMENT: Patients were recruited from those referred to the CNR-JGH clinic for consultation by their treating oncology teams at the Jewish General Hospital: an urban university teaching hospital in Montréal, Québec, Canada. The participants recruited included both those with impaired performance status (PS = 2 or 3) [20] and patients with better performance who were unable to attend hospital-based supervised physical rehabilitation more than once a week due to travel distance or other similar constraints. Patients were excluded if they had any cognitive impairment affecting their ability to apply NMES safely without medical supervision, skin conditions contraindicating repeated use of surface electrodes, orthopedic implants in the hip joint, metastatic lesions to the femur, or electronic implants (e.g., pacemakers and defibrillators). Patients who were already actively participating in a supervised rehabilitation or exercise intervention greater than once a week were also ineligible. BODY.METHODS.BASELINE FUNCTIONAL ASSESSMENT: All assessments, instruction and follow-ups were performed by the study physiotherapist in the CNR-JGH clinic. Baseline functional evaluation was performed as follows: a) Walking endurance capacity was assessed with the 6-minute walk test (6 MWT) [21]. If patients were unable to walk independently even with standard walking devices (including rolling walking frames) a score of 0 m was recorded. b) Lower extremity functional strength was assessed using the repeated Sit-to-Stand (STS) test, as included in the Simmonds Functional Assessment Battery [22]. The patient was instructed to go from sitting to standing twice in a row as quickly as possible, without the use of their arms. The test was performed twice and the average time of the two trials was recorded. c) Global functional performance status (PS) [20] was recorded based on patient's report of their level of physical activity to the study physiotherapist. BODY.METHODS.INSTRUCTION AND USE OF NEUROMUSCULAR ELECTRICAL STIMULATION (NMES) MACHINE: Participants were instructed on the application of the surface electrodes and the use the portable NMES machine (Neurotech MediStim, Galway, Ireland). Initial training was performed to ensure that participants were able to apply the electrodes and use the device on themselves without additional assistance. After cleaning the skin with an alcohol pad, disposable electrodes (EMPI Self-adhesive Carbon FM electrodes 2"x 2") were applied to the motor points of the Vastus Medialis Oblique distally, and over the midpoint of the quadriceps muscle belly more proximally [23]. The electrodes placed on each thigh were then connected to the individual leads of the NMES unit, to allow for separate intensity control for each quadriceps. Participants were instructed to use NMES at home for 6 weeks on both quadriceps muscles simultaneously for 30 minutes a day. Stimulation parameters were as follows: 300 ms pulses, 50 Hz, alternating 5 s on 5 s off (50% duty). Stimulation intensity was individually adjusted for each limb in order to obtain tetanic contraction or maximum tolerated intensity. In addition, participants were instructed to voluntarily contract the quadriceps muscles during the periods of NMES stimulation (active co-contraction) to enhance the strengthening effect and improve tolerance of NMES. Participants were positioned with their legs on a horizontal surface, and the knee supported on a rolled towel. They were asked to perform an isometric contraction of the quadriceps for the duration of the stimulus, by tightening the thigh muscle and pushing the knee down on towel without lifting the foot. Where appropriate, participants could progress the co-contraction exercise if their strength improved. For this second stage they were instructed to raise their leg off the surface and completely extend the knee against gravity. BODY.METHODS.STUDY COMPLETION AND ADHERENCE WITH NMES INTERVENTION: The study physiotherapist called or met with each participant at the mid-point (3 weeks) of the study to give additional instruction if needed. Each patient had a maximum of 6 weeks (42 days) of NMES and the total (days) and % maximum possible adherence were calculated using the logs of daily NMES use completed by each patient. Study completion was achieved if patients completed the assessment at the end of the intervention period. Patient adherence was measured using an intervention diary in which participants were asked to log their use of the NMES machine daily, and document any variance from the prescribed intervention. The percentage of maximum possible adherence was calculated using the total number of days during the intervention period when the patient could have used the NMES machine. Thus, if the patient was hospitalized for 7 days during the study, the % maximum possible adherence was calculated as a proportion of 35 days not 42. BODY.METHODS.OUTCOME ASSESSMENTS.PRIMARY OUTCOMES: Feasibility was assessed by measuring the proportion of patients who completed the 6-week intervention, and by the overall level of adherence achieved over the intervention period. Acceptability of NMES was evaluated at the end of the 6 weeks by a brief structured questionnaire developed for this study which was completed by each patient. The questionnaire used a 5-point Likert scale to rate their answers from 1 = "strongly disagree", 2 = "disagree", 3 = "neutral", 4 = "agree", 5 = "strongly agree". BODY.METHODS.OUTCOME ASSESSMENTS.SECONDARY OUTCOMES: Physical performance measures were performed at baseline (PS, 6 MWT, STS) and at the end of the study. To determine the effects of adherence with NMES on physical function parameters, results were compared in patients with <40% (poor) vs >40% NMES adherence. BODY.METHODS.ETHICS: The study was approved by the Jewish General Hospital Research Ethics Committee and all participants provided written informed consent. BODY.METHODS.STATISTICAL ANALYSIS: Comparison between participants who did and did not complete the study was performed using unpaired t-test and Fisher's exact test for continuous and categorical variables respectively. Assessment of change from before and after NMES intervention was performed using paired t-tests or Fisher's exact test for PS category count data. To assess factors potentially relevant to adherence and change in physical performance tests, mean differences were compared using t-tests and differences in counts within categories were assessed using Fisher's exact test. All analyses were performed using R [24]. BODY.RESULTS: 111 new patients were seen in the CNR-JGH clinic between June 2010 and July 2011. Of these 52 (47%) were potentially eligible for the study (poor performance status and/or inability to attend hospital-based exercise training). After further screening, 37 patients were approached and of these 18 (49%) patients were recruited. The reasons that 19 patients were not consented for the study ranged from medical deterioration (3) or improvement (1), preference for standard exercise (4), feeling that the study was not of interest or too burdensome (6), and follow-up and communication difficulties with study coordinator (5). Of the 18 patients who gave signed consent, three withdrew from the study and did not proceed to baseline assessment (Figure 1). Reasons for early withdrawals included: rapid medical deterioration and death (n = 1), started exercising regularly at a local gym (n = 1), withdrawn due to language and communication difficulties which became evident only after consent was obtained (n = 1). The remaining 15 patients underwent baseline function testing and their demographic and disease characteristics are shown in Table 1. Two patients with good PS (PS = 1) were also included in the study as they were not exercising regularly and could not attend the usual hospital-based supervised training sessions. The majority of patients (73%) had metastatic (stage IV) disease. The most common tumour types were lung or gastrointestinal cancers, but five patients had other types of tumours including breast (n = 1), ovarian (n = 1), endometrial (n = 1) and haematological (n = 1) malignancy. One additional patient was being treated with cyclical intravenous chemotherapy for systemic amyloidosis without overt evidence of underlying malignancy. Overall 67% of the patients were undergoing active treatment with chemotherapy and 40% were taking, or had completed within the preceding 14 days, an extended (≥3 weeks' duration) course of corticosteroids (Table 1). Results of functional testing at baseline are shown in Table 2. As a group, the patients in this study had impaired exercise capacity with mean 6 MWT of 257 m (50.2% predicted) and mean STS time of 8.0 s, which is 2.6 standard deviations above the mean expected in (i.e. slower than) healthy individuals of similar age (Table 2). Figure 1Breakdown of reasons for early and late withdrawals from the NMES study. Table 1 Demographic and disease characteristics of patients who underwent full baseline assessment     All subjects (N = 15)       Completed (N = 10) Withdrawn (N = 5)     Mean (SD)     P Age yrs 67.9 (9.4) 67.6 (10.9) 68.4 (6.3) 0.88 Body mass index kg/m 2 23.1 (4.6) 23.7(4.9) 22.1(4.2) 0.52   N (%)       Sex  M 9 (60) 4 (40) 5 (100)    F 6 (40) 6 (60) 0 (0) 0.04 PS  1 2 (13) 2 (20) 0 (0)    2 6 (40) 4 (40) 2 (40)    3 7 (47) 4 (40) 3 (60) 0.79 Diagnosis  Lung cancer 4 (27) 3 (30) 1 (20)    GI cancer 6 (40) 2 (20) 4 (80)    Other 5 (33) 5 (50) 0 (0) NA Cancer stage  III 1 (7) 1 (10) 0 (0)    IV 11 (73) 6 (60) 5 (100)    NA 3 (20) 3 (30) 0 (0) NA Chemotherapy  Y 10 (67) 6 (60) 4 (80)    N 5 (33) 4 (40) 1 (20) 0.60 Recent steroid use  Y 6 (40) 4 (40) 2 (40)    N 9 (60) 6 (60) 3 (60) 1.00 Notes: P indicates result of significance testing (unpaired t-test for means or Fisher’s exact test for count data) comparing patients who did, or did not, complete the study. Table 2 Physical performance evaluation results at baseline and after six weeks of NMES intervention         Baseline End of study         All subjects (N = 15)         Withdrawn (N = 5) Completed (N = 10) P a Completed (N = 10) Difference P b   Number     PS 1     0 2 0.79 # 4   0.15 #   2     2 4   4       3     3 4   2       Mean (SD)   6 MWT m   257 (160) 166 (138) 303 (157) 0.11 282 (171) −21.1 (167.7) 0.70   %   50.2 (32.2) 31.0 (24.6) 59.8 (32.2) 0.08 56.0 (34.5) −3.8 (33.3) 0.73 STS s   8.0 (4.3) 7.6 (3.4) 8.2 (4.8) 0.80 7.0 (3.3) −1.2 (3.4) 0.30   S-score   2.6 (2.4) 2.2 (2.0) 2.7 (2.2) 0.69 2.0 (1.8) −0.7 (2.0) 0.30 Notes: Performance status (PS), six-minute walk distance (6 MWT) in metres (m) and expressed as % predicted (from [ 21 ]), Sit-to-stand (STS) test expressed as seconds (s) and as a standard score (S-score) value calculated using age range-specific mean and standard deviations for healthy controls (from [ 22 ]): positive scores indicates STS S-scores above the mean. P a: # Fisher’s exact (for counts) or unpaired t-test (for means) comparing baseline results for patients withdrawn and patients who completed study. P b : # Fisher’s exact (for counts) or paired t-test (for means) comparing baseline and final test results for patients who completed the study. BODY.RESULTS.FEASIBILITY: Completion of the study was achieved by 10 (66.6%) of the 15 patients. All 15 had baseline assessments, used the NMES for the 6-week intervention period, and completed the final assessments. Four of the five late withdrawals were not due to the NMES intervention, namely: death (n = 1) and deterioration in medical status (n = 3). Only one patient withdrew because they found they could not tolerate the local discomfort due to repeated NMES treatments (Figure 1). For those completing the study the machine was used for mean(SD) 20.1(13.0) days during the 42-day study period. When the adherence was adjusted to account for periods when NMES had to be stopped during hospital admissions, this showed that patients used their machines over half the available time (mean(SD) 54.2(32.8)%). On closer examination there were in fact three groups: three poorly adherent patients (mean(SD) use 14.3(6.3)%), three moderately adherent patients (mean(SD) use 49.4(1.1)%) and four highly adherent patients (mean(SD) use 87.8(8.7)%) (Figure 2). Thus, 7(70%) patients completing the study achieved more than 40% adherence: equivalent to NMES use at least three times a week. In subsequent analysis to explore the potential impact of adherence on physical function two groups were defined: poorly adherent (<40%, n = 3) and adherent (>40%, n = 7) patients. Figure 2Histogram of adherence with NMES intervention during study. BODY.RESULTS.ACCEPTABILITY: Informal feedback during the study was positive about the use of NMES at home. Furthermore, the acceptability of NMES at a group level was captured using the questionnaires at the end of the study. More than 50% of patients felt strongly that 6 weeks' of NMES use was acceptable (Table 3: C). Furthermore, a majority of patients reported that NMES was a useful in helping with activities and symptoms (Table 3: F, G). In addition 50% of patients were interested in using NMES outside of a study (Table 3: H). Table 3 Responses to feedback questions for patients completing the study   Question text Median Range A “NMES was helpful for me” 4 2-5 B “NMES is something patients can be taught to use safely at home” 5 3-5 C “Using the NMES for 6 weeks is acceptable” 5 2-5 D “Using the NMES for 30 mins a day is acceptable” 4 2-5 E “Home NMES was a good intervention for me” 4 2-5 F “Using NMES had a positive impact on my daily activities” 4 2-5 G “Using NMES had a positive impact on my symptoms” 4 2-5 H “If offered outside of a study, I would want to use NMES” 3 2-5 Notes: Allowed responses ranged from 1–5 where 5 = “totally agree” (see Methods). BODY.RESULTS.CHANGES IN PHYSICAL FUNCTION: Baseline and final assessment results were compared for Completers (Table 2). Global performance status and STS appeared somewhat better, but these changes did not achieve statistical significance (Fisher's exact test p = 0.15 comparing categories of PS; mean reduction in STS = −1.2s, P = 0.3). Only one patient was unable to perform the 6 MWT at baseline, but they had improved and walked 220m at end of study. However, as a group there was no change in mean 6 MWT distance after the NMES intervention period (mean difference = −21.1m, P = 0.7) (Table 2). Adherence with NMES might be expected to be a major determinant of improvement in physical function, but we were not able to detect any such effect of adherence with the number of patients recruited in this study. Thus, the patients with poor adherence (<40%, n = 3) did not have significantly different changes in STS or 6 MWT compared to the adherent patients (>40%, n = 7) (P = 0.56, 0.71 respectively). It was also noted that those on long term steroids had significantly reduced 6 MWT at beginning of study (mean(SD): recent steroids 166(139)m vs no steroids 394(88)m, P = 0.04). We hypothesized that NMES may have a particularly useful role in promoting improving function in these patients. However despite a trend towards differential improvement in patients on steroids, this did not reach statistical significance (6 MWT mean(SD) change: recent steroids 35(184)m, no steroids −59(161)m, P = 0.4). A test of proportions gave similar suggestive but non-significant results for both change in 6 MWT and PS, e.g. for PS: 3 out of 4 patients who had recently been on an extended course of oral corticosteroids reported improved PS compared with only 1 out of 6 non-steroid-treated patients (Fisher's exact test, P = 0.19). BODY.DISCUSSION: This is the first report of the use of NMES in patients with advanced cancer and poor performance status or other barriers to usual exercise training. Our results suggest that NMES is feasible and well-tolerated in this group of patients with advanced cancer who are unable to attend hospital-based exercise training. Only one patient (7%) withdrew from the study because they were unable to tolerate the NMES intervention, whereas four patients (27%) withdrew or could not complete the study, because of medical complications or death. Furthermore, qualitative feedback confirmed that home-based NMES is highly acceptable. There was a wide range in adherence with NMES over the 6-week period of intervention, but despite this, 70% of patients used the NMES for at least 40% of the available days. This equates to use at least three times a week, which is the usual frequency for standard exercise training programs. Repeated physical performance testing before and after NMES revealed wide intra-individual variability. However, there was no improvement in objective measurements of exercise function after NMES intervention (Table 2). Furthermore, no dose-effect of NMES was demonstrated, as there were no differences in physical functional outcomes, between adherent (>40% maximum usage) and poorly adherent (<40% maximum NMES usage) patients. We recognize a number of limitations to interpreting our results, including the small number of analyzable patients (10) in this pilot study. The primary study objective was to assess feasibility and acceptability of NMES in a variety of cancer patients and for this reason the patients recruited for the study had a range of pathological types and stages of cancer. However, as a result the study participants also had a widely varying initial functional status and exercise performance, making it more difficult to reliably detect functional changes attributable to NMES. We did not include an untreated control group to assess the likely spontaneous changes in outcome measures over the same time period. In debilitated advanced cancer patients, progressive physical deterioration may be anticipated. Thus a reasonable minimal expectation of successful NMES, and other exercise interventions, would be to inhibit this deterioration, rather than achieve an improvement from baseline. Without an untreated comparison group we are unable to say whether there was any indication of this beneficial effect of NMES in the current study. Due to limited personnel resources, the same physiotherapist who instructed the patient in the use of NMES also performed the outcomes assessments. This raises the possibility of patient response bias, particularly for reported global performance status and responses to questionnaire items. Maddocks et al. [18] reported only 15/30 lung cancer patients receiving palliative chemotherapy completed approximately 10 week NMES intervention. In contrast to the present study, they concluded that NMES was not an acceptable treatment based on a pre-defined 80% group adherence threshold [18]. However it is notable that in line with our results only 3(11%) of their patients withdrew because of NMES discomfort suggesting that the intervention, per se, is tolerable for the majority. Furthermore, without any correction for periods when patients were hospitalized etc., 50% of patients in the study by Maddocks et al. achieved the required three times a week treatment frequency, and another 3 patients fell below this threshold because of only one missed session. We would concur with the authors of that study who acknowledged that their conclusion, that NMES is "unacceptable", may have been based on unrealistically stringent criteria [18]. Published studies of use of NMES describe a range of different electrical stimulation protocols [25] and there is no clear consensus as to the optimal stimulation frequency, electrode placement sites and duty-cycle etc. The original intention in this study was to use dual stimulation of quadriceps and gluteus muscles. However, it became clear that there were both practical and patient acceptability difficulties in repeatedly and consistently placing the gluteus muscle electrodes, especially in very sedentary patients or those requiring help from caregivers to do so. As a result a simplified protocol was used with electrical stimulation of quadriceps only, combined with active voluntary co-contraction of the stimulated limb muscles, to enhance the contractile stimulus. The use of active co-contraction is novel and proved both practical and acceptable as demonstrated by the feedback received (Table 3). Other informal comments received also suggested the patients appreciated the ability to participate in their exercise training even in this limited way. Our study was not powered to detect differences in the physical outcome measures and many other confounding factors, including acute changes in medical status, can influence the results of the physical performance tests. The physical outcomes measures adopted (PS, STS, 6 MWT) have been widely used in studies of cancer patients and are relevant to normal daily functional activities [22]. However, direct measurements of muscle strength or cross-sectional area, have been used by other authors [11,14,17], and these approaches may be more sensitive to detect modest short-term improvements in muscle induced by NMES. Analysis of pooled data from several different studies of NMES in chronic medical conditions suggests NMES use leads to a mean improvement in 6 MWT of 40 m [26]. However we calculated that 127 patients would be needed to detect a difference of 40 m in 6 MWT (effect size 0.25 SD) with 80% power in any future study, without modifying the entry criteria. This is directly related to the wide variance in initial 6 MWT results. Thus stratifying analysis or selecting patients with a pre-defined baseline performance (e.g. by 6 MWT distance) would make it easier to detect subsequent changes after NMES intervention. High drop-rate rates and the continuing uncertainty about which patients are most likely to respond to NMES physiologically, present significant challenges to successful completion of studies of NMES in cancer patients. It is noteworthy that many studies in COPD and CHF conclude that use of NMES may be best targeted to the most debilitated patients with very poor function [14]. However cancer patients who have both advanced disease and impaired functional status (e.g. 6 MWT <350 m at baseline [27]) have very poor prognosis, and even short-term studies in these patients are likely to suffer substantial dropouts [3,8] due to medical deterioration and death. In this pilot study we were not able to confidently identify any subgroups of patients who have greater potential to complete the NMES intervention. Thus patients who did not complete the intervention had a trend towards lower mean baseline 6 MWT distance (166 m Withdrawn vs 303 m Completed, P = 0.11) (Table 2). However, neither this nor any other demographic or physical function results at baseline were significantly different between those that completed and those who were withdrawn from the study (Tables 1, 2). One group of patients who may be more likely to benefit from NMES are those that have taken a recent prolonged course of corticosteroids, given that corticosteroids are both widely used in cancer therapy and can cause myopathy [28]. Consistent with this notion, the data from this study did confirm that those patients recently treated with steroids had poorer function at baseline (6 MWT mean(SD): recent steroids 166(139)m vs no steroids 394(88)m, P = 0.04). However there was only a non-significant trend towards a differential improvement in 6 MWT in patients treated with extended courses of steroids. Nevertheless, further studies of NMES may be warranted to establish if early use of NMES can be of particular benefit in cancer patients treated with prolonged courses of steroids. Similarly, it is conceivable that other subgroups of cancer patients should be considered for more focused studies, including the more elderly patients with comorbidities [29] and those being treated with antitumour agents, such as Sorafenib, which promote muscle loss [30]. BODY.CONCLUSIONS: The results of the current pilot study suggest that NMES is both feasible and acceptable in a mixed group of patients with cancer, most of whom had poor performance status. This study does not demonstrate that NMES leads to improved physical functioning in cancer patients with poor performance status despite the success of NMES in other chronic diseases and clinical settings. One reason for the difficulty in detecting functional benefits of NMES in cancer patients may be their marked heterogeneity in terms of medical status and physical functioning. As a result, though it is likely that NMES has a role in achieving or maintaining optimal functional ability in selected patients with cancer, this still remains to be confirmed by future prospective studies. Such studies should be designed to both identify the best candidates for NMES interventions and to confirm the optimal method of delivery of this type of therapeutic exercise. BODY.ABBREVIATIONS: 6MWT: Six-minute walk test; CHF: Congestive heart failure; CNR-JGH: The McGill Cancer Nutrition-Rehabilitation Program clinic at the Jewish General Hospital; COPD: Chronic obstructive pulmonary disease; NMES: Neuromuscular electrical stimulation; PS: Performance status; STS: Sit-to-stand test. BODY.COMPETING INTERESTS: RTJ has received payments as an advisory board member for NEOMED and lecturer fees from Abbott Laboratories. The authors confirm that individuals or groups who have provided funding for the CNR-JGH activities have not been involved in any aspect of the study design, execution or analysis. BODY.AUTHORS’ CONTRIBUTIONS: RTJ and TS conceived and designed the study, wrote the protocol and obtained initial ethics approval. TW finalized the study protocol, performed the patient testing and supervised the NMES interventions. BV recruited patients and coordinated the study including completion of study database. RTJ analysed the study results. All authors contributed to the writing of the manuscript. All authors read and approved the final manuscript. BODY.AUTHORS’ INFORMATION: TS and TW are physiotherapists who worked in the CNR-JGH team during the study. BV worked as study coordinator with the CNR-JGH team during the study. RTJ is an MD and Director of the CNR-JGH team. BODY.PRE-PUBLICATION HISTORY: The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1472-684X/13/23/prepub

TITLE: Resistant starch lowers postprandial glucose and leptin in overweight adults consuming a moderate-to-high-fat diet: a randomized-controlled trial ABSTRACT.BACKGROUND: High-amylose maize resistant starch type 2 (HAM-RS2) stimulates gut-derived satiety peptides and reduces adiposity in animals. Human studies have not supported these findings despite improvements in glucose homeostasis and insulin sensitivity after HAM-RS2 intake which can lower adiposity-related disease risk. The primary objective of this study was to evaluate the impact of HAM-RS2 consumption on blood glucose homeostasis in overweight, healthy adults. We also examined changes in biomarkers of satiety (glucagon-like peptide-1 [GLP-1], peptide YY [PYY], and leptin) and body composition determined by anthropometrics and dual-energy x-ray absorptiometry, dietary intake, and subjective satiety measured by a visual analogue scale following HAM-RS2 consumption. ABSTRACT.METHODS: Using a randomized-controlled, parallel-arm, double-blind design, 18 overweight, healthy adults consumed either muffins enriched with 30 g HAM-RS2 (n = 11) or 0 g HAM-RS2 (control; n = 7) daily for 6 weeks. The HAM-RS2 and control muffins were similar in total calories and available carbohydrate. ABSTRACT.RESULTS: At baseline, total PYY concentrations were significantly higher 120 min following the consumption of study muffins in the HAM-RS2 group than control group (P = 0.043). Within the HAM-RS2 group, the area under the curve (AUC) glucose (P = 0.028), AUC leptin (P = 0.022), and postprandial 120-min leptin (P = 0.028) decreased independent of changes in body composition or overall energy intake at the end of 6 weeks. Fasting total PYY increased (P = 0.033) in the HAM-RS2 group, but changes in insulin or total GLP-1 were not observed. Mean overall change in subjective satiety score did not correlate with mean AUC biomarker changes suggesting the satiety peptides did not elicit a satiation response or change in overall total caloric intake. The metabolic response from HAM-RS2 occurred despite the habitual intake of a moderate-to-high-fat diet (mean range 34.5% to 39.4% of total calories). ABSTRACT.CONCLUSION: Consuming 30 g HAM-RS2 daily for 6 weeks can improve glucose homeostasis, lower leptin concentrations, and increase fasting PYY in healthy overweight adults without impacting body composition and may aid in the prevention of chronic disease. However, between-group differences in biomarkers were not observed and future research is warranted before specific recommendations can be made. ABSTRACT.TRIAL REGISTRATION: None. BODY.INTRODUCTION: Epidemiological observations show that consuming a diet high in fiber can lower the risk for obesity, obesity-related comorbidities, and reduce all-cause mortality [1, 2]. One systematic review that examined the effects of dietary fiber on body weight reported that a 0.4% reduction in body weight can be achieved by consuming most dietary fibers for 4 weeks [3]. However, the amount of weight lost was dependent on the physiochemical properties (solubility, fermentability, and viscosity) of each type of fiber [3]. The purported mechanisms by which fiber contributes to weight loss such as altering gut motility, attenuating nutrient absorption, and lowering overall caloric intake are also associated with the physiochemical properties [2, 4]. Fermentable fibers are receiving attention because the metabolites produced from bacterial fermentation in the gastrointestinal (GI) tract can influence body weight. These fibers produce short chain fatty acids (SCFA; acetate, propionate, butyrate) in the distal intestine that stimulate the release of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) that act synergistically with leptin, an adipokine primarily released from adipose tissue, to induce satiety and regulate energy expenditure through central nervous system actions [5–7]. Despite increased SCFA production from fiber fermentation, the relationship between GLP-1 and PYY on satiety and food intake in humans is inconsistent. After consuming a standardized breakfast on the morning immediately following 3 days of consecutive intake of a barley kernel-based bread with resistant starch, fasting plasma GLP-1 and postprandial PYY concentrations increased in healthy middle-aged adults [8]. However, changes in appetite sensations (satiety, hunger, and desire to eat) did not occur [8]. Similarly, overweight women did not elicit a postprandial subjective satiety response despite improvements of GLP-1 and PYY after consuming an enzyme-hydrolyzed arabinoxylan from wheat or intact arabinoxylan from flax at breakfast [9]. In contrast, in healthy adults, the upregulation of GLP-1 and PYY corresponded with enhanced subjective satiety immediately after resistant maltodextrin intake [10]. However, subsequent energy intake was not suppressed despite these improvements. One recent trial found improvements in the area under the curve (AUC) PYY that corresponded with satiety and a 14% reduction in food intake in healthy adults consuming 15 g unripe banana flour rich in resistant starch for 6 weeks [11]. The different satiety peptides and satiation responses may be related to fermentability patterns, the type, amount, and duration of fiber intake, and the gut microbiota composition of individuals. Also, blood gut peptides may be too low in concentration to cross the blood brain barrier or the individuals may have hypothalamic resistance, which can occur from a high-fat diet even in lieu of obesity [12]. High-amylose maize resistant starch type 2 (HAM-RS2) is an insoluble, nonviscous fermentable fiber that has been shown to improve glucose homeostasis and/or peripheral insulin sensitivity among individuals who were healthy with normal glucose homeostasis [13] or those with metabolic syndrome [14–16]. However, many of these trials of longer duration did not report or show improvements in blood concentrations of gut peptides, satiety responses, or changes in food intake. HAM-RS2 may exert its benefits on glucose metabolism by increasing SCFA in the blood to alter free fatty acid and glycerol release from adipocytes and increased fat oxidation [17], modulate bile acid metabolism [18], or alter the gut microbiota profile [19]. Most previous trials reported the impact of HAM-RS2 on glucose homeostasis in either healthy individuals or those with metabolic syndrome [12–15]. Therefore, our primary aim was to determine the impact of the daily consumption of 30 g HAM-RS2 incorporated into muffins for 6 weeks on glucose homeostasis in normoglycemic, healthy overweight adults at risk for developing glycemic abnormalities. We also measured fasting and postprandial biomarker concentrations known to influence satiety (GLP-1, PYY, and leptin), subjective satiety, dietary intake, and body composition in these individuals. BODY.MATERIALS AND METHODS.PARTICIPANTS: Healthy overweight adults with a body mass index (BMI) ≥ 28 kg/m2 between 18 and 50 years of age of any race or ethnicity were recruited from Denton, Texas and the surrounding area. Participants were sedentary (<20 min of activity no more than 2 days per week) for at least 6 months prior to enrollment in the study. Exclusion criteria included those diagnosed with or taking medication(s) for chronic conditions, such as diabetes, hypertension, cancer, diseases of the liver, kidney, or heart, or other metabolic disorders. Participants were also excluded if they gained or lost a significant amount of weight or followed a special diet ≤ 3 months prior to enrollment, consumed vitamins, minerals, or antioxidants in excess of amounts found in a daily multivitamin tablet, or dietary supplements known to alter metabolism, had an intolerance to the study foods, or smoked. Women who were pregnant, lactating, or interested in becoming pregnant were not eligible. The study was approved by the Institutional Review Board at Texas Woman's University. All participants provided written consent prior to study enrollment. BODY.MATERIALS AND METHODS.PROTOCOL: The study was a randomized-controlled, parallel-arm, double-blind design lasting 6 weeks. Individuals who met the screening criteria and agreed to participate in the study were randomized to either the HAM-RS2 group or control group using a random numbers generator from SPSS version 19 (IBM Corporation, Armonk, NY, USA). Prior to participant randomization, the study muffins were formulated at the University food preparation laboratory. The treatment muffins were developed to provide 50 g Hi-Maize® 260 resistant starch (~30 g HAM-RS2, ~20 g equal mixture slowly and rapidly digestible starch; Ingredion Incorporated, Westchester, IL, USA) daily. The control muffins (0 g HAM-RS2) were developed to contain similar amounts of available carbohydrate and minimal differences in total calories than the treatment muffins (Table 1). Each 60 g treatment muffin contained 16.7 g Hi-Maize® 260 resistant starch to provide 10 g HAM-RS2, therefore three muffins (180 g cooked) were required to obtain 30 g HAM-RS2 daily as indicated in the protocol. No difference in overall muffin likeability was found based on sensory evaluations of HAM-RS2 and control muffins using a 9-point hedonic scale prior to the implementation of this study [20]. All study muffins were prepared, packaged, and labeled by culinary and nutrition students twice per week to ensure freshness and quality. Two flavors of the treatment and control muffins were developed, pumpkin spice and cranberry spice. The different flavors of muffins were administered on alternating weeks to alleviate monotony and improve compliance. The two flavors of muffins were closely matched for total calories and macronutrients. The cranberry spice muffins included 4.3 g dried cranberries, but no pumpkin puree. The pumpkin spice muffins included 6.8 g pumpkin puree without dried cranberries. Different amounts of dried cranberries and pumpkin puree were added so the caloric value of each muffin type was similar.Table 1Comparison of nutrients between muffinsa NutrientHAM-RS2ControlEnergy (kcal)b 349.6389.1Available carbohydrate (g)c 70.571.6Protein (g)5.410.6Fat (g)5.16.6Fiber (g)30.911.4 aNutrient analysis based on the amount consumed daily by each participant; 180 g cooked (baked) weight. Analysis conducted by Pope Labs, Irving, Texas using the following Official AOAC Methods: moisture 925.10; oil 923.03; ash 923.03; nitrogen 988.05A; total dietary fiber 991.43 bEnergy does not include contribution from short chain fatty acids associated with fermentation cAvailable carbohydrate excludes fiber The participants arrived for baseline data collection following an overnight fast. Anthropometric measurements were obtained in triplicate followed by the baseline blood collection (time 0). Participants consumed all three of their respective study muffins within 15 min along with 6 oz of orange juice. Immediately following muffin intake the participants completed a visual analogue scale (VAS). Four additional postprandial blood samples were collected at 15, 30, 60, and 120 min. Nutrition education and body composition analysis occurred between blood collections. Participants were instructed by a Registered Dietitian Nutritionist (RDN) to follow a balanced diet according to the Dietary Guidelines for Americans 2010 [21] adjusted for the caloric value of the study muffins and to remain sedentary during the study. Body composition was determined by whole body dual-energy x-ray absorptiometry (DXA) using the Lunar DPX NT model (GE Healthcare, Fairfield, CT, USA). Body composition analysis and blood collections were repeated using the same protocol as baseline at the end of the intervention (week 6). BODY.MATERIALS AND METHODS.DIETARY INTAKE AND BOWEL HABITS: Three days prior to baseline measurements, at midpoint (week 3), and the end of the study (week 6) all participants completed bowel habit logs and dietary intake journals for 3 days as instructed by a RDN. Dietary intake was analyzed using the United States Department of Agriculture National Nutrient Database for Standard Reference [22]. The bowel habit logs evaluated tolerance and potential adverse events from study muffin consumption by asking a series of questions adapted from Lewis et al. [23], such as "describe the consistency of your stools," and provided a space for comments. Participants were instructed to contact investigators immediately if abnormal changes in bowel habits, or the presence of blood or mucus, were observed. Muffin intake compliance was assessed through food intake journals. BODY.MATERIALS AND METHODS.SUBJECTIVE SATIETY MEASUREMENTS: Each VAS was 100 mm in length with questions at each end to indicate feelings of subjective satiety ranging from "not at all" to "very much or a lot". The 7 questions were adapted from Flint et al. [24] and included "how hungry are you?", "how satisfied do you feel?", "how full do you feel?", "how much do you think you can eat?", "how pleasant would you find eating another mouthful of this food?" "would you like to eat something sweet?", and "would you like to eat something fatty?". BODY.MATERIALS AND METHODS.BIOMARKERS: A phlebotomist drew approximately 12 mL of blood at each time point into EDTA vacutainers (BD Diagnostics, Franklin Lakes, NF, USA). The tubes were centrifuged at 3,200 rpm for 12 min at 4 °C. Plasma was immediately aliquoted into 1.5 mL Eppendorf microcentrifuge tubes and stored at −80 °C until analyzed for glucose, insulin, GLP-1, PYY, and leptin. Glucose was determined using a hexokinase colorimetric method (Stanbio Laboratory, Boerne, TX, USA) with an intra-assay coefficient of variation (CV) <6%. Total insulin (CV < 8%; Alpco, Salem, NH, USA), leptin (CV < 6%; Raybiotech, Norcross, GA, USA), and total PYY (PYY(1–36) and PYY (3–36); CV < 6%; EMD Millipore, Billerica, MA, USA) were measured using an enzyme-linked immunosorbent (ELISA) technique. Total GLP-1 (GLP-1(7–36) and GLP-1 (9–36); CV < 7%; Raybiotech, Norcross, GA, USA) concentrations were determined by an enzyme immunoassay protocol. BODY.MATERIALS AND METHODS.STATISTICAL ANALYSIS: To assess differences between and within the HAM-RS2 and control groups, change from mean baseline and final scores were calculated for anthropometric, body composition, satiety (after three questions were reverse coded), and biomarker concentrations from each individual blood collection time points (fasting, 15, 30, 60, and 120 min). Due to limited sample size and potential deviations from normality, primary analyses were conducted using nonparametric tests. The Wilcoxon Signed Ranked test compared differences within groups, while the Mann–Whitney U test examined between group differences. The total area under the curve (AUC) was calculated for all plasma biomarkers using the trapazoidal rule and was compared using the nonparametric tests described above. Pearson's correlation coefficient examined associations between dependent outcomes. Data are presented as mean ± standard error of the mean (SEM), unless otherwise noted. SPSS version 19 (IBM Corporation, Armonk, NY, USA) and statistical significance was achieved with a P-value ≤ 0.05. BODY.RESULTS.PARTICIPANTS: Twenty-five participants were enrolled in the study; however, only 18 (83% female) completed the protocol and were included in the data analysis (Fig. 1). Baseline characteristics of the participants who completed the study and were included in the data analysis did not differ between groups (Table 2).Fig. 1Consort Diagram Table 2Baseline participant characteristicsCharacteristicHAM-RS2 (n = 11)Control (n = 7) P valuea Female, n (%)9 (81.8)6 (85.7)Age, years31.0 ± 3.031.2 ± 4.20.973Body mass index (kg/m2)34.8 ± 1.530.6 ± 1.50.085Waist circumference (cm)99.1 ± 3.791.6 ± 3.30.151Total body fat mass (kg)b 42.8 ± 3.134.6 ± 3.00.085Total body lean mass (kg)b 47.3 ± 1.843.7 ± 2.30.179Trunk total mass (kg)b 47.1 ± 3.643.9 ± 6.00.285Trunk fat mass (kg)b 22.2 ± 2.119.8 ± 3.00.285Trunk lean mass (kg)b 23.8 ± 1.823.1 ± 2.90.479Android fat (kg)b 50.8 ± 2.150.1 ± 0.80.791Gynoid fat (kg)b 52.2 ± 2.649.6 ± 1.00.285Visceral adipose tissue (in3)b 76.8 ± 13.963.0 ± 18.10.417Data presented as mean ± SEM except for qualitative values. All body composition measurements were obtained in triplicate after fasting for ≥8 h aBetween group differences determined by Mann–Whitney U test bMeasured by dual-energy x-ray absorptiometry (Lunar DPX NT model, GE Healthcare, Fairfield, CT, USA) BODY.RESULTS.DIETARY INTAKE AND COMPLIANCE: Energy and macronutrient (carbohydrate, protein, lipid) intake were similar between groups throughout the study (Table 3). Mean daily fiber intake significantly increased by 113% at midpoint in the HAM-RS2 group. At week 6, the mean fiber intake in the HAM-RS2 group increased 100% from baseline indicating fiber intake was sustained throughout the study. In contrast, the control group had a significant reduction in both fiber and carbohydrate intake from midpoint to the end of the study. Dietary fiber intake was considerably higher in the HAM-RS2 group at midpoint and the end of the study than the control. An unexplained reduction in dietary carbohydrate and fiber intake occurred from week 3 to week 6 in the control group. At baseline the HAM-RS2 group consumed a high fat diet averaging 95.8 ± 29.5 g/d (39.3% of total calories), which was non-significantly reduced to 82.1 ± 25.1 g/d (34.8% of total calories) at week 6. Based on data from the food intake journals, the HAM-RS2 and control groups consumed 94 and 98% of the study muffins, respectively, at midpoint suggesting high compliance. Compliance decreased to 85% in the HAM-RS2 group and 73% in the control group at the end of the study.Table 3Changes in mean macronutrient intake between HAM-RS2 and control groups1,2 NutrientHAM-RS2Control P value** BaselineWeek 3Week 6BaselineWeek 3Week 6Calories2190 ± 5642193 ± 3972123 ± 4242091.1 ± 455.32046. ± 13371910.5 ± 648.70.256Carbohydrate (g)257.5 ± 84.8283.0 ± 73.2268.7 ± 62.6294.3 ± 90.4289.4 ± 173.7a 261.7 ± 84.0a 1.000Protein (g)82.7 ± 15.378.0 ± 13.278.4 ± 24.788.0 ± 11.174.8 ± 48.367.4 ± 32.70.462Lipid (g)95.8 ± 29.582.5 ± 20.782.1 ± 25.175.5 ± 23.970.1 ± 52.470.8 ± 27.00.462Fiber (g)21.5 ± 13.9a,b 45.7 ± 8.21* 43.1 ± 6.7b 29.5 ± 14.530.2 ± 18.0a* 23.8 ± 6.8a 0.001 1Dietary analysis software used is United States Department of Agriculture (USDA) National Nutrient Database for Standard Reference (http://ndb.nal.usda.gov) 2Within group values with the same letter superscript (a,b) in the same row are statistically different (P < 0.05) *Between group values are statistically different (P < 0.05) by Mann–Whitney U test **Between group comparisons at week 6 analyzed by Mann–Whitney U test BODY.RESULTS.TOLERANCE TO STUDY MUFFINS: In both groups, stool consistency was relatively stable throughout the study. No differences in reaching the bathroom in time, usage of toilet paper, or marks on undergarments occurred across groups at any time point. One participant in the HAM-RS2 group recorded the presence of blood or mucus in stool on two separate occasions; one prior to receiving the intervention and the other at midpoint. This participant was 89% compliant with muffin consumption at midpoint based on food records suggesting intake remained adequate. Overall, the HAM-RS2 and control muffins were well tolerated and few changes in bowel habit indicators were documented throughout the study. BODY.RESULTS.SUBJECTIVE SATIETY: Mean overall satiety score did not differ within or between groups (P = 0.230). The mean score for the question "how full do you feel?" in the HAM-RS2 group increased from baseline to the end of the study and approached significance (P = 0.058) (data not shown). BODY.RESULTS.BODY COMPOSITION: Consuming muffins with HAM-RS2 did not significantly change body composition. A decrease (P = 0.043) in total trunk mass and a near significant decrease in lean trunk mass (P = 0.063) occurred in the control group (data not shown). At baseline the HAM-RS2 group had higher total leg mass (P = 0.011) than the control group, and the difference was sustained at the end of the intervention (P = 0.02) (data not shown). BODY.RESULTS.BIOMARKERS: Changes from baseline to week 6 in the AUC for the plasma biomarkers are shown in Table 4. At the end of the 6-week treatment, the AUC change from baseline was not significantly different between HAM-RS2 and control groups for any biomarker measures. However, a significant within-group decrease in both AUC glucose (P = 0.028) and AUC leptin (P = 0.022) was observed from baseline to week 6 in the HAM-RS2 group. There were no changes from baseline to the end of the intervention in the AUC for any biomarker in the control group. Within-group comparisons for biomarkers collected at each time point at the end of the intervention are shown in Fig. 2. Only one biomarker differed between groups when examining single blood collection time points. The 120 min postprandial concentration of PYY (P = 0.043) was higher in the HAM-RS2 group than the control group at baseline. Within-group differences in leptin and PYY were found from baseline to week 6 in the HAM-RS2 group. A decrease in postprandial leptin (P = 0.028) occurred at 120 min in the HAM-RS2 group. A within-group change in PYY was also observed in the HAM-RS2 group where fasting concentrations increased (P = 0.033) from baseline to the end of the intervention. Within the control group, no differences in the individual time points or AUC for any biomarker occurred.Table 4Mean AUC biomarker comparisons within and between groupsBiomarkersHAM-RS2Control P value1 Change from baseline to week 6 P value2 Change from baseline to week 6 P value2 Glucose−1588 ± 5450.028−790 ± 7060.3100.285Insulin−908 ± 9410.333518 ± 11971.0000.425GLP-1120 ± 940.139208 ± 1270.1760.791PYY438 ± 7190.285954 ± 14650.8660.085Leptin−664 ± 2350.022−191 ± 2300.3980.425Data presented as mean ± SEM 1Between group comparisons at week 6 by Mann–Whitney U test 2Within group comparisons from baseline to week 6 by Wilcoxon Signed-Ranked test Fig. 2Change in biomarker concentrations from baseline to week 6 in the HAM-RS2 and control groups. This figure shows within-group comparisons from Wilcoxon Signed Ranked tests and between-group comparisons from Mann–Whitney U tests. aIndicates significant within-group changes in biomarkers from baseline to week 6. Fasting PYY increased (P = 0.033) while leptin decreased (P = 0.028) 120 min after study foods were consumed in the HAM-RS2 group. A near-significant decrease (P = 0.062) in leptin also occurred 60 min after study foods were consumed in the HAM-RS2 group. bIndicates significant difference at baseline between groups in 120 min postprandial PYY concentrations (P = 0.043) BODY.RESULTS.RELATIONSHIP BETWEEN SUBJECTIVE SATIETY AND BIOMARKERS OF SATIETY: Correlations between the mean AUC for each biomarker and the mean score for each VAS question were not found in either the control or HAM-RS2 group at the end of the intervention; however, correlations between body composition measurements and the AUC for several biomarkers were found. In the HAM-RS2 group, BMI (r = 0.655; P = 0.029), percent total fat (r = .889; P < 0.001), total trunk mass (r = 0.851; P = 0.001); trunk fat (r = 0.700; P = 0.017); trunk lean (r = 0.795; P = 0.003) were associated with the AUC glucose. The percent total body fat correlated (r = 0.652; P = 0.030) with AUC leptin. In the control group AUC glucose was associated with BMI (r = 0.814; P = 0.026), total fat (r = 0.801; P = 0.030), percent fat (r = 0.879; P = 0.009), and percent trunk fat (r = 0.772; P = 0.042). Total trunk lean mass correlated with the AUC insulin (r = 0.792; P = 0.034) in the control group. The AUC glucose was associated with the AUC insulin in both the HAM-RS2 (r = 0.710; P = 0.014) and control (r = 0.785; P = 0.036) groups. BODY.DISCUSSION: Our primary aim was to examine changes in glucose homeostasis after consuming 30 g HAM-RS2 for 6 weeks in overweight adults. We also measured the plasma biomarkers (GLP-1, PYY, and leptin) and subjective satiety which could alter dietary intake and body composition. We found significant reductions in AUC glucose and AUC leptin in the HAM-RS2 group although differences between groups did not occur. In addition, a significant increase in fasting PYY occurred within the HAM-RS2 group after consuming the treatment muffins for 6 weeks. Interestingly, the favorable changes in biomarkers in the HAM-RS2 group did not elicit changes in overall mean subjective satiety score or body composition at the end of the intervention. Only one biomarker differed between groups throughout the duration of the study. Baseline PYY 120-min post-muffin intake was significantly higher in the HAM-RS2 group which may be attributed to initial HAM-RS2 fermentation. Increasing the duration of the. intervention or sample size may have produced additional between-group changes in biomarkers. The decrease in AUC glucose in the HAM-RS2 group occurred under normoglycemic conditions and no change in overall mean carbohydrate intake suggesting other contributing mechanisms. One mechanism could be due to the SCFA produced from the fermentation of HAM-RS2 by bacteria in the lower GI tract. Butyrate and propionate are substrates for intestinal gluconeogenesis [25]. The newly synthesized glucose from the intestine reduces overall hepatic gluconeogenesis through portal vein sensors that contribute to overall blood glucose control [25]. Interestingly, HAM-RS2 lowered glucose AUC in the presence of a high-fat diet. At baseline habitual dietary fat intake in the HAM-RS2 group was 39.4% of total calories (~95 g per day). It is well established that diets high in fat consisting of large amounts of saturated and omega-6 polyunsaturated fatty acids and lower omega-3 polyunsaturated fatty acids contribute to chronic inflammation [26] and the development of chronic disease. Interestingly when dietary composition contains only 30.4% of calories from fat, HAM-RS2 can suppress inflammation and normalize glucose by mediating gluconeogenesis potential and altering hepatic fuel usage from lipids to carbohydrate [27]. Despite observing an improvement in AUC glucose in the HAM-RS2 group, we did not see changes in plasma insulin or insulin sensitivity (determined by Homeostasis Model Assessment estimates) that have been reported in human trials [13–15]. We did, however, observe a positive correlation between AUC glucose and AUC insulin in both groups. A novel finding from our study is that AUC leptin significantly decreased from baseline to the end of the intervention in the HAM-RS2 group independent of changes in body composition. We also found a significant postprandial reduction at 120 min and near significant postprandial reductions at 30 (P = 0.074) and 60 min (P = 0.062) in leptin. Leptin is primarily produced by adipocytes and blood concentrations correlate with adipocyte size and percent body fat. One plausible mechanism could be enhanced fat oxidation which has been observed in healthy adults where postprandial fat oxidation increased 23% after 5.4% of dietary carbohydrate, but not 10.7%, was consumed acutely as HAM-RS2 [17]. Approximately 11% of mean daily carbohydrate intake was in the form of HAM-RS2 in our study; however, our population and study duration differed from Higgins et al. [17]. Another study found increased fat oxidation when resistant starch type 4 (RS4) plus whey protein was administered to healthy overweight and lean women [28]. Increased fat oxidation and resting energy expenditure also occurred in healthy lean males following consumption of 38 g RS4 in a mixed meal [29]. In addition, a fermentable cereal fiber reduced leptin by enhancing the gene expression of several enzymes involved in fat oxidation [30]. Similar to our findings, So et al. [31] reported lower leptin in addition to smaller adipocyte size in mice consuming HAM-RS2 compared to mice on a low resistant starch diet even though body composition did not differ between the groups. In contrast, no change in adipose leptin mRNA expression or plasma leptin concentrations occurred after a meal tolerance test in healthy males who consumed 30 g HAM-RS2 over 4 weeks [13]. The study by Robertson et al. examined healthy individuals with an average BMI of 23.7 kg/m2 which is much lower than our mean baseline BMI of 34.8 kg/m2, and our intervention was longer in duration. This suggests that duration of HAM-RS2 consumption and degree of adiposity may also be important in modulating leptin. Differences in adiposity (mean total body fat mass) between HAM-RS2 and control groups (P = 0.085) may explain why leptin did not differ between groups in our study, or that changes in leptin are more sensitive in individuals with higher fat mass. The reduction in leptin after HAM-RS2 intake may benefit individuals with leptin resistance. Leptin resistance can develop from consuming high fat diets [12, 32] and sustained elevated leptin concentrations [33]. In our study, the HAM-RS2 group had a mean fasting leptin concentration of 33 ± 6 ng/mL. Fasting leptin concentrations of ≥15 ng/mL have been described as the cutoff value to predict insulin resistance [34]. Although we did not observe insulin resistance in the HAM-RS2 group, it is plausible that the study participants in our study were resistant to leptin due to high blood leptin concentrations and consumption of a high-fat diet at baseline. We observed significant reductions in leptin, but also a non-significant mean reduction (5.5%) in total calories from fat, but not overall total calories, in the HAM-RS2 group. This decrease in dietary fat is not likely responsible for the decrease in AUC leptin concentrations [35]. Similar to our results, leptin decreased with the addition of a fermentable soluble fiber in obese rats ingesting a high-fat diet [36]. Interestingly, the decrease in leptin in this study did not induce an orexigenic effect since overall caloric intake did not change and was similar to the control. Our study also found an increase in total fasting PYY but not postprandial or AUC PYY in the HAM-RS2 group at the end of the study. The increase may be related to the carry-over fermentation effects of prior-day HAM-RS2 intake. Increased PYY has been observed with fermentable fiber consumption in animal studies [37]. PYY binds to the Y2 receptors of the arcuate nucleus to elicit a satiation response alongside increased energy expenditure [38]. Two isoforms of PYY exist: PYY1–36 and PYY3–36. PYY1–36 predominates under fasting conditions and has a lower affinity to the Y2 receptor. This may explain why we did not see a relationship between total PYY and satiety. At baseline, PYY was significantly higher (P = 0.043) in the HAM-RS2 group than control 120 min after the consumption of the study muffins and may be due to the initial fermentation of HAM-RS2 following intake. A similar increase in PYY occurred in healthy adults consuming resistant starch and whey protein 180 min after intake [28]. We did not observe improvements in GLP-1, which is consistent with several human studies examining HAM-RS2 intake in overweight adults for ≥4 weeks [39, 40]. This study has several limitations. First, only inactive concentrations of GLP-1 and total PYY (PYY(1–36) + PYY(3–36)) were measured. We were not able to determine the exact PYY isoform which may have explained why subjective satiety did not change. Another limitation includes the lack of adding di-peptidyl peptidase-4 inhibitors (DPP-IV) following blood collection. DPP-IV rapidly degrades GLP-1, thus we were not able to measure the physiologically active form of GLP-1 [41]. Also, the participants consumed the study muffins at any time during the day. Consuming the muffins at one meal or equally distributed throughout the day could impact metabolic response. In addition, the participants consumed the study muffins prior to postprandial blood collection instead of an isocaloric mixed meal equal in macronutrient composition. Thus, the plasma biomarker response is reflective of study muffin consumption. However, a between group 120-min improvement in PYY was observed in the HAM-RS2 group indicating fermentation can upregulate this satiety peptide. Lastly, a significant reduction in daily fiber intake from midpoint to week 6 occurred in the control group indicating the control muffins replaced other high-fiber foods in the diet. BODY.CONCLUSION: In conclusion, daily consumption of 30 g of HAM-RS2 in muffins over 6 weeks can decrease leptin concentrations, assist with blood glucose homeostasis, and improve fasting PYY in healthy overweight adults. These findings occurred without changes in total caloric intake or body composition. Adding HAM-RS2 to the diet can improve fiber intake to enhance overall diet quality. However, strong conclusions cannot be made due to small sample size and between group differences in biomarkers were not observed at the end of the intervention. The mechanisms associated with biomarker changes in the HAM-RS2 group are likely related to the fermentation of HAM-RS2 by gut microbiota, but additional research is needed to determine the type, amount, and duration of resistant starch that would provide the most advantageous physiological results.